Production of isoprenoids

ABSTRACT

A recombinant yeast that makes an isoprenoid compound is provided. The yeast comprises an endogenous mevalonate pathway comprising (i) an enzyme that converts acetyl-CoA to acetoacetyl-CoA, (ii) an enzyme that converts acetoacetyl-CoA to hydroxymethylglutaryl-CoA, (iii) an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid, (iv) an enzyme that converts mevalonic acid to phosphomevalonic acid, (v) an enzyme that converts phosphomevalonic acid to phosphomevalonate, and (vi) an enzyme that converts phosphomevalonate to isopentenyl pyrophosphate. The yeast further comprises heterologous nucleic acid sequences encoding an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid, an enzyme that converts mevalonic acid to phosphomevalonic acid, an enzyme that converts phosphomevalonic acid to phosphomevalonate, and an enzyme that converts phosphomevalonate to isopentenyl pyrophosphate. Carbon flow through the mevalonate pathway is increased in the recombinant yeast compared to a yeast that does not comprise the heterologous nucleic acid molecule encoding an enzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/089,267, filed Apr. 18, 2011, which is a continuation of U.S. patentapplication Ser. No. 12/942,809, filed Nov. 9, 2010, now U.S. Pat. No.7,927,862, which is a continuation of U.S. patent application Ser. No.11/753,301, filed May 24, 2007, now U.S. Pat. No. 7,838,279, which is acontinuation of U.S. patent application Ser. No. 10/719,196, filed Nov.20, 2003, which is a continuation of U.S. patent application Ser. No.09/909,558, filed Jul. 20, 2001, now U.S. Pat. No. 6,689,593, which is acontinuation of U.S. patent application Ser. No. 09/350,275, filed Jul.6, 1999, now U.S. Pat. No. 6,531,303, which claims priority to U.S.Provisional Patent Application No. 60/091,964, filed Jul. 6, 1998. Eachof these priority documents is incorporated herein, in their entirety,by this reference.

FIELD OF THE INVENTION

The present invention relates to the biological production of farnesoland geranylgeraniol.

BACKGROUND OF THE INVENTION

Geranylgeraniol and farnesol are synthesized as, inter alia, precursorsof cholesterol and are used as highly lipophilic molecules for fixatingproteins to cell membranes. In addition, geranylgeraniol and farnesolexhibit wide range of biological activities such as antimicrobial,antiviral and antitumor activities. These compounds also possess abilityto prevent a wide variety of illnesses such as ulcer, neuro-degenerativeillnesses, conditions linked to skin aging, the phenomena of thrombosisand atherosclerosis and immune deficiencies. In addition,geranylgeraniol diphosphate is a precursor for the biological synthesisof TAXOL®, which is a potent anticancer compound currently marketed byBristol-Myers Squibb Co. under the generic name paclitaxel.

There remains a need for an efficient and economical method forproducing farnesol and/or geranylgeraniol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate a diagramatic representation of themevalonate-dependent isoprenoid biosynthetic pathway.

FIGS. 1C-1D illustrate a diagramatic representation of themevalonate-independent isoprenoid biosynthetic pathway.

FIG. 2 illustrates the growth and farnesol production of strains derivedfrom strain MBNA1-13.

FIG. 3 illustrates the growth of wild-type, erg9 and repaired ERG9strains.

FIG. 4 illustrates the production of farnesol in microorganisms withamplified production of HMG CoA reductase.

FIG. 5 illustrates the production of farnesol and GG in strainsoverexpressing GGPP synthases.

FIG. 6 illustrates the effect of amplified production of FPP synthase onfarnesol and GG production.

FIGS. 7A-7B illustrate the pathway for metabolic conversion of isoprenoland prenol to farnesol.

DETAILED DESCRIPTION OF THE INVENTION 1.0 Introduction

The present invention provides a method for producing farnesol andgeranylgeraniol (GG). The present invention also includes variousaspects of biological materials and intermediates useful in theproduction of farnesol and GG by biological production.

Isoprenoids are the largest family of natural products, with about22,000 different structures known. All isoprenoids are derived from theC₅ compound isopentylpyrophosphate (IPP). Thus, the carbon skeletons ofall isoprenoid compounds are created by sequential additions of the C₅units to the growing polyprenoid chain.

While the biosynthetic steps leading from IPP to isoprenoids areuniversal, two different pathways leading to IPP exist. Fungi (such asyeast) and animals possess the well known mevalonate-dependent pathway(depicted in FIGS. 1A-1B) which uses acetyl CoA as the initialprecursor. Bacteria and higher plants, on the other hand, possess anewly discovered mevalonate independent pathway, also referred to hereinas the non-mevalonate pathway, (depicted in FIGS. 1C-1D) leading frompyruvate and glyceraldehyde 3-phosphate [Lois et al., Proc. Natl. Acad.Sci. USA, 95, 2105-2110 (1998); Rohmer et al., J. Am. Chem. Soc. 118,2564-2566 (1996); Arigoni, et al., Proc. Natl. Acad. Sci. USA, 94,10600-10605 (1997); Lange et al., Proc. Natl. Acad. Sci. USA, 95,2100-2104 (1998)]. In plants, there is evidence that both themevalonate-dependent and -independent pathways exist, the former beingcytosolic and the latter being plastidial [Arigoni, et al., Proc. Natl.Acad. Sci. USA, 94, 10600-10605 (1997); Lange et al., Proc. Natl. Acad.Sci. USA, 95, 2100-2104 (1998)]. Several steps of themevalonate-independent pathway have been established. The first step,catalyzed by D-1-deoxyxylulose 5-phosphate synthase, formsD-1-deoxyxylulose 5-phosphate from pyruvate and glyceraldehyde3-phosphate. The second and third steps, catalyzed by D-1-deoxyxylulose5-phosphate reductoisomerase, catalyze the conversion ofD-1-deoxyxylulose 5-phosphate to 2-C-methyl D erythritol-4-P (MEP).Several further reactions are required to convert MEP to IPP, and theseenzymes are unknown at this time [Lois et al., Proc. Natl. Acad. Sci.USA, 95, 2105-2110 (1998); Takahashi et al., Proc. Natl. Acad. Sci. USA,95, 2100-2104 (1998); Duvold et al. Tetrahedron Letters, 38, 4769-4772(1997)].

Farnesol and GG are prenyl alcohols produced by dephosphorylation offarnesylpryrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP),respectively. FPP and GGPP are intermediates in the biosynthesis ofisoprenoid compounds, including sterols, ubiquinones, heme, dolichols,and carotenoids, and are used in the post-translational prenylation ofproteins. Both FPP and GGPP are derived from IPP. Embodiments of thepresent invention include the biological production of farnesol or GG inprokaryotic or eukaryotic cell cultures and cell-free systems,irrespective of whether the organism utilizes the mevalonate-dependentor -independent pathway for the biosynthesis of the precursor of allisoprenoids, IPP. Reference herein to farnesyl phosphate orgeranylgeranyl phosphate refers to the respective mono-, di- andtri-phosphate compounds, unless one specific form is specificallydesignated.

2.0 Modified Microorganism

Suitable biological systems for producing farnesol and GG includeprokaryotic and eukaryotic cell cultures and cell-free systems.Preferred biological systems include fungal, bacterial and microalgalsystems. More preferred biological systems are fungal cell cultures,more preferably a yeast cell culture, and most preferably aSaccharomyces cerevisiae cell culture. Fungi are preferred since theyhave a long history of use in industrial processes and can bemanipulated by both classical microbiological and genetic engineeringtechniques. Yeast, in particular, are well-characterized genetically.Indeed, the entire genome of S. cerevisiae has been sequenced, and thegenes coding for enzymes in the isoprenoid pathway have already beencloned. Also, S. cerevisiae grows to high cell densities, and amounts ofsqualene and ergosterol (see FIG. 1A) up to 16% of cell dry weight havebeen reported in genetically-engineered strains. For a recent review ofthe isoprenoid pathway in yeast, see Parks and Casey, Annu. Rev.Microbiol. 49:95-116 (1995).

The preferred prokaryote is E. coli. E. coli is well established as anindustrial microorganism used in the production of metabolites (aminoacids, vitamins) and several recombinant proteins. The entire E. coligenome has also been sequenced, and the genetic systems are highlydeveloped. As mentioned above, E. coli uses the mevalonate-independentpathway for synthesis of IPP. The E. coli dxs, dxr, idi, and ispA genes,encoding D-1-deoxyxylulose 5-phosphate synthase, D-1-deoxyxylulose5-phosphate reductoisomerase, IPP isomerase, and FPP synthase,respectively, have been cloned and sequenced [Fujisaki, et. al, J.Biochem. 108, 995-1000 (1990); Lois et al., Proc. Natl. Acad. Sci. USA,95, 2105-2110 (1998); Hemmi et al., J. Biochem., 123, 1088-1096 (1998)].

Preferred microalga for use in the present invention include Chlorellaand Prototheca.

Suitable organisms useful in producing farnesol and GG are availablefrom numerous sources, such as the American Type Culture Collection(ATCC), Rockville, Md., Culture Collection of Algae (UTEX), Austin,Tex., the Northern Regional Research Laboratory (NRRL), Peoria, Ill. andthe E. coli Genetic Stock Center (CGSC), New Haven, Conn. In particular,there are culture collections of S. cerevisiae that have been used tostudy the isoprenoid pathway which are available from, e.g., Jasper Rineat the University of California, Berkeley, Calif. and from Leo Parks atNorth Carolina State University, Raleigh, N.C.

Preferably the cells used in the cell culture are genetically modifiedto increase the yield of farnesol or GG. Cells may be geneticallymodified by genetic engineering techniques (i.e., recombinanttechnology), classical microbiological techniques, or a combination ofsuch techniques and can also include naturally occurring geneticvariants. Some of such techniques are generally disclosed, for example,in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Labs Press. The reference Sambrook et al., ibid., isincorporated by reference herein in its entirety. A genetically modifiedmicroorganism can include a microorganism in which nucleic acidmolecules have been inserted, deleted or modified (i.e., mutated; e.g.,by insertion, deletion, substitution, and/or inversion of nucleotides),in such a manner that such modifications provide the desired effect ofincreased yields of farnesol or GG within the microorganism or in theculture supernatant. As used herein, genetic modifications which resultin a decrease in gene expression, in the function of the gene, or in thefunction of the gene product (i.e., the protein encoded by the gene) canbe referred to as inactivation (complete or partial), deletion,interruption, blockage or down-regulation of a gene. For example, agenetic modification in a gene which results in a decrease in thefunction of the protein encoded by such gene, can be the result of acomplete deletion of the gene (i.e., the gene does not exist, andtherefore the protein does not exist), a mutation in the gene whichresults in incomplete or no translation of the protein (e.g., theprotein is not expressed), or a mutation in the gene which decreases orabolishes the natural function of the protein (e.g., a protein isexpressed which has decreased or no enzymatic activity). Geneticmodifications which result in an increase in gene expression or functioncan be referred to as amplification, overproduction, overexpression,activation, enhancement, addition, or up-regulation of a gene. Additionof cloned genes to increase gene expression can include maintaining thecloned gene(s) on replicating plasmids or integrating the cloned gene(s)into the genome of the production organism. Furthermore, increasing theexpression of desired cloned genes can include operatively linking thecloned gene(s) to native or heterologous transcriptional controlelements.

2.1 Squalene Synthase Modifications

Embodiments of the present invention include biological production offarnesol or GG by culturing a microorganism, preferably yeast, which hasbeen genetically modified to modulate the activity of one or more of theenzymes in its isoprenoid biosynthetic pathway. In one embodiment, amicroorganism has been genetically modified by decreasing (includingeliminating) the action of squalene synthase activity (see FIG. 1). Forinstance, yeast erg9 mutants that are unable to convert mevalonate intosqualene, and which accumulate farnesol, have been produced. Karst andLacroute, Molec. Gen. Genet., 154, 269-277 (1977); U.S. Pat. No.5,589,372. As used herein, reference to erg9 mutant or mutationgenerally refers to a genetic modification that decreases the action ofsqualene synthase, such as by blocking or reducing the production ofsqualene synthase, reducing squalene synthase activity, or inhibitingthe activity of squalene synthase, which results in the accumulation offarnesyl diphosphate (FPP) unless the FPP is otherwise converted toanother compound, such as farnesol by phosphatase activity. Blocking orreducing the production of squalene synthase can include placing theERG9 gene under the control of a promoter that requires the presence ofan inducing compound in the growth medium. By establishing conditionssuch that the inducer becomes depleted from the medium, the expressionof ERG9 (and therefore, squalene synthase synthesis) could be turnedoff. Also, some promoters are turned off by the presence of a repressingcompound. For example, the promoters from the yeast CTR3 or CTR1 genescan be repressed by addition of copper. Blocking or reducing theactivity of squalene synthase could also include using an excisiontechnology approach similar to that described in U.S. Pat. No.4,743,546, incorporated herein by reference. In this approach, the ERG9gene is cloned between specific genetic sequences that allow specific,controlled excision of the ERG9 gene from the genome. Excision could beprompted by, for example, a shift in the cultivation temperature of theculture, as in U.S. Pat. No. 4,743,546, or by some other physical ornutritional signal. Such a genetic modification includes any type ofmodification and specifically includes modifications made by recombinanttechnology and by classical mutagenesis. Inhibitors of squalene synthaseare known (see U.S. Pat. No. 4,871,721 and the references cited in U.S.Pat. No. 5,475,029) and can be added to cell cultures. In anotherembodiment, an organism having the mevalonate-independent pathway ofisoprenoid biosynthesis (such as E. coli) is genetically modified sothat it accumulates FPP and/or farnesol. For example, decreasing theactivity of octaprenyl pyrophosphate synthase (the product of the ispBgene) would be expected to result in FPP accumulation in E. coli. (Asai,et al., Biochem. Biophys. Res. Comm. 202, 340-345 (1994)). The action ofa phosphatase could further result in farnesol accumulation in E. coli.

Yeast strains need ergosterol for cell membrane fluidity, so mutantsblocked in the ergosterol pathway, such as erg9 mutants, need extraneousergosterol or other sterols added to the medium for the cells to remainviable. The cells normally cannot utilize this additional sterol unlessgrown under anaerobic conditions. Therefore, a further embodiment of thepresent invention is the use of a yeast in which the action of squalenesynthase is reduced, such as an erg9 mutant, and which takes upexogenously supplied sterols under aerobic conditions. Geneticmodifications which allow yeast to utilize sterols under aerobicconditions are demonstrated below in the Examples section (seeExample 1) and are also known in the art. For example, such geneticmodifications include upc (uptake control mutation which allows cells totake up sterols under aerobic conditions) and hem1 (the HEM1 geneencodes aminolevulinic acid synthase which is the first committed stepto the heme biosynthetic pathway from FPP, and hem1 mutants are capableof taking up ergosterol under aerobic conditions following a disruptionin the ergosterol biosynthetic pathway, provided the cultures aresupplemented with unsaturated fatty acids). Yeast strains having thesemutations can be produced using known techniques and also are availablefrom, e.g., Dr. Leo Parks, North Carolina State University, Raleigh,N.C. Haploid cells containing these mutations can be used to generateother mutants by genetic crosses with other haploid cells. Also,overexpression of the SUT1 (sterol uptake) gene can be used to allow foruptake of sterols under aerobic conditions. The SUT1 gene has beencloned and sequenced. Bourot and Karst, Gene, 165: 97-102 (1995).

In a further embodiment, microorganisms of the present invention can beused to produce farnesol and/or GG by culturing microorganisms in thepresence of a squalene synthase inhibitor. In this manner, the action ofsqualene synthase is reduced. Squalene synthase inhibitors are known tothose skilled in the art. (See, for example, U.S. Pat. No. 5,556,990.

2.2 HMG-CoA Reductase Modifications

A further embodiment of the present invention is the use of amicroorganism which has been genetically modified to increase the actionof HMG-CoA reductase. It should be noted that reference to increasingthe action of HMG-CoA reductase and other enzymes discussed hereinrefers to any genetic modification in the microorganism in questionwhich results in increased functionality of the enzymes and includeshigher activity of the enzymes, reduced inhibition or degradation of theenzymes and overexpression of the enzymes. For example, gene copy numbercan be increased, expression levels can be increased by use of apromoter that gives higher levels of expression than that of the nativepromoter, or a gene can be altered by genetic engineering or classicalmutagenesis to increase the activity of an enzyme. One of the keyenzymes in the mevalonate-dependent isoprenoid biosynthetic pathway isHMG-CoA reductase which catalyzes the reduction of3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). This is the primaryrate-limiting and first irreversible step in the pathway, and increasingHMG-CoA reductase activity leads to higher yields of squalene andergosterol in a wild-type strain of S. cerevisiae, and farnesol in anerg9 strain. One mechanism by which the action of HMG-CoA reductase canbe increased is by reducing inhibition of the enzyme, by eithergenetically modifying the enzyme or by modifying the system to removethe inhibitor. For instance, both sterol and non-sterol products of theisoprenoid pathway feedback inhibit this enzyme (see, e.g., Parks andCasey, Annu. Rev. Microbiol. 49:95-116 (1995). Alternatively or inaddition, the gene(s) coding for HMG-CoA reductase can be altered bygenetic engineering or classical mutagenesis techniques to decrease orprevent inhibition. Also, the action of HMG-CoA reductase can beincreased by increasing the gene copy number, by increasing the level ofexpression of the HMG-CoA reductase gene(s), or by altering the HMG-CoAreductase gene(s) by genetic engineering or classical mutagenesis toincrease the activity of the enzyme. See U.S. Pat. No. 5,460,949, theentire contents of which are incorporated herein by reference. Forexample, truncated HMG-CoA reductases have been produced in which theregulatory domain has been removed and the use of gene copy numbers upto about six also gives increased activity. Id. See also, Downing etal., Biochem. Biophys. Res. Commun., 94, 974-79 (1980) describing twoyeast mutants having increased levels of HMG-CoA reductase. Two isozymesof HMGCoA reductase, encoded by the HMG1 and HMG2 genes, exist in S.cerevisiae. The activity of these two isozymes is regulated by severalmechanisms including regulation of transcription, regulation oftranslation, and for Hmg2p, degradation of the enzyme in the endoplasmicreticulum (Hampton and Rine, 1994; Donald, et. al. 1997). In both Hmg1pand Hmg2p, the catalytic domain resides in the —COOH terminal portion ofthe enzyme, while the regulatory domain resides in the membrane spanningNH₂-terminal region. It has been shown that overexpression of just thecatalytic domain of Hmg1p in S. cerevisiae increases carbon flow throughthe isoprenoid pathway, resulting in overproduction of squalene(Saunders, et. al. 1995; Donald, et. al., 1997). The present inventorshave expressed the catalytic domain of the S. cerevisiae Hmg2p instrains having a normal (i.e., unblocked) isoprenoid pathway andobserved a significant increase in the production of squalene.Furthermore, overexpression of the catalytic domain of Hmg2p resulted inincreased farnesol production in an erg9 mutant, and increased farnesoland GG production in an erg9 mutant overexpressing GGPP synthase, grownin fermentors.

2.3 GGPP Synthase Modifications

A further embodiment of the present invention is the use of amicroorganism which has been genetically modified to increase the actionof GGPP synthase. Genes coding for this enzyme from a variety ofsources, including bacteria, fungi, plants, mammals, and archaebacteria,have been identified. See, Brinkhaus et al., Arch. Biochem. Biophys.,266, 607-612 (1988); Carattoli et al., J. Biol. Chem., 266, 5854-59(1991); Chen et al., J. Biol. Chem., 268, 11002-11007 (1993); Dogbo etal., Biochim. Biophys. Acta, 920, 140-148 (1987); Jiang et al., J. Biol.Chem., 270, 21793-99 (1995); Kuntz al., Plant J., 2, 25-34 (1992);Laferriere, et al., Biochim. Biophys. Acta, 1077, 167-72 (1991); Math etal., Proc. Natl. Acad. Sci. USA, 89, 6761-64 (1992); Ohnuma et al., J.Biol. Chem., 269, 14792-97 (1994); Sagami et al., Arch. Biochem.Biophys., 297, 314-20 (1992); Sagami et al., J. Biol. Chem., 269,20561-66 (1994); Sandmann et al., J. Photochem. Photobiol. B: Biol., 18,245-51 (1993); Scolnik et al., Plant Physiol., 104, 1469-70 (1994);Tachibana et al., Biosci. Biotech. Biochem., 7, 1129-33 (1993);Tachibana et al., J. Biochem., 114, 389-92 (1993); Wiedemann et al.,Arch. Biochem. Biophys., 306, 152-57 (1993). Some organisms have abifunctional enzyme which also serves as an FPP synthase, so it isinvolved in the overall conversion of IPP and DMAPP to FPP to GGPP (seeFIG. 1). Some enzymes, such as those found in plants, have relaxedspecificity, converting IPP and DMAPP to GGPP (see FIG. 1). Geneticmodifications of GGPP synthase, as used herein, encompass engineering amonofunctional GGPP synthase or a bifunctional FPP/GGPP synthase toenhance the GGPP synthase activity component of the enzyme. A preferredGGPP synthase gene is the BTS1 gene from S. cervisiae. The BTS1 gene andits isolation are described in Jiang et al., J. Biol. Chem., 270,21793-99 (1995) and copending application Ser. No. 08/761,344, filed onDec. 6, 1996, now issued as U.S. Pat. No. 5,912,154, the completedisclosure of which incorporated herein by reference. However, GGPPsynthases of other hosts can be used, and the use of the bifunctionalGGPP synthases may be particularly advantageous in terms of channelingcarbon flow through FPP to GGPP, thereby avoiding loss of FPP tocompeting reactions in the cell.

In further embodiments of the invention, in addition to themodifications of GGPP synthase described above, the wild type GGPPsynthase is eliminated from the production organism. This would serve,for example, to eliminate competition between the modified GGPP synthaseand the wild type enzyme for the substrates, FPP and IPP. Deletion ofthe wild-type gene encoding GGPP synthase could also improve thestability of the cloned GGPP synthase gene by removing regions of highgenetic sequence homology, thereby avoiding potentially detrimentalgenetic recombination.

2.4 FPP Synthase Modifications

A further embodiment of the present invention is the use of amicroorganism which has been genetically modified to increase the actionof FPP synthase.

Genes coding for this enzyme from a variety of sources have beenidentified. See, Anderson et al., J. Biol. Chem., 264, 19176-19184(1989); Attucci, et al., Arch. Biochem. Biophys., 321, 493-500 (1995);Cane et al., J. Am. Chem. Soc., 105, 122-124 (1983); Chambon et al.,Current Genetics, 18, 41-46 (1990); Chambon et al., Lipids, 26, 633-36(1991); Chen al., Protein Science, 3, 600-607 (1994); Davisson, et al.,J. Am. Chem. Soc., 115, 1235-45 (1993); Ding et al., Biochem. J., 275,61-65 (1991); Hugueney et al., FEBS Letters, 273, 235-38 (1990); Joly etal., J. Biol. Chem., 268, 26983-89 (1993); Koyama al., J. Biochem., 113,355-63 (1993); Sheares, et al., Biochem., 28, 8129-35 (1989); Song etal., Proc. Natl. Acad. Sci. USA, 91, 3044-48 (1994); Spear et al., J.Biol. Chem., 267, 14662-69 (1992); Spear et al., J. Biol. Chem., 269,25212-18 (1994). Anderson et al., J. Biol. Chem., 264, 19176-19184(1989) reported a 2-3 fold overexpression of FPP synthase with the S.cerevisiae gene in a yeast shuttle vector.

As described in the Examples section, it has been surprisingly foundthat overexpression of FPP synthase did not lead to an increase infarnesol production, but unexpectedly lead to an increase in theproduction of GG in the absence of any overexpression of GGPP synthase.

In further embodiments of the invention, in addition to themodifications of FPP synthase described above, the wild type FPPsynthase is eliminated from the production organism. This would serve,for example, to eliminate competition between the modified FPP synthaseand the wild type enzyme for the substrates, IPP, DMAPP and GPP.Deletion of the wild-type gene encoding FPP synthase could also improvethe stability of the cloned FPP synthase gene by removing regions ofhigh genetic sequence homology, thereby avoiding potentially detrimentalgenetic recombination.

2.5 Phosphatase Modifications

A further embodiment of the present invention is the use of amicroorganism which has been genetically modified to increasephosphatase action to increase conversion of FPP to farnesol or GGPP toGG. For example, both S. cerevisiae and E. coli contain numerousphosphatase activities. By testing several phosphatases for efficientdephosphorylation of FPP or GGPP, one could select an appropriatephosphatase and express the gene encoding this enzyme in a productionorganism to enhance farnesol or GG production. In addition to (orinstead of) increasing the action of a desired phosphatase to enhancefarnesol or GG production, one could eliminate, through genetic means,undesirable phosphatase activities. For example, one could eliminatethrough mutation the activity of a phosphatase that specifically acts onFPP, so that the FPP that was spared would be available for conversionto GGPP and subsequently GG.

2.6 Additional Genetic Modifications.

Modifications of Other Isoprenoid Pathway Enzymes.

Modifications that can be made to increase the action of HMGCoAreductase, GGPP synthase and phosphatases are described above.Modification of the action of isoprenoid pathway enzymes is not limitedto those specific examples, and similar strategies can be applied tomodify the action of other isoprenoid pathway enzymes such asacetoacetyl Co-A thiolase, HMG-CoA synthase, mevalonate kinase,phosphomevalonate kinase, phosphomevalonate decarboxylase, IPPisomerase, farnesyl pyrophosphate synthase or D-1-deoxyxylulose5-phosphate synthase D-1-deoxyxylulose 5-phosphate reductoisomerase.

Engineering of Central Metabolism to Increase Precursor Supply to theIsoprenoid Pathway.

In organisms having the mevalonate-dependent isoprenoid pathway, thebiosynthesis of farnesol or GG begins with acetyl CoA (refer to FIG. 1).One embodiment of the present invention is genetic modification of theproduction organism such that the intracellular level of acetyl CoA isincreased, thereby making more acetyl CoA available for direction to theisoprenoid pathway (and hence to farnesol and/or GG). For example, thesupply to acetyl CoA can be increased by increasing the action of thepyruvate dehydrogenase complex. The supply of acetyl CoA can be furtherincreased by increasing the level of pyruvate in the cell by increasingthe action of pyruvate kinase. In organisms having themevalonate-independent isoprenoid pathway, the biosynthesis ofisoprenoids begins with pyruvate and glyceraldehyde 3-phosphate. Thesupply of pyruvate and glyceraldehyde 3-phosphate available forisoprenoid biosynthesis can be increased by increasing the action ofpyruvate kinase and triophosphate isomerase, respectively.

The examples above are provided only to illustrate the concept ofengineering central metabolism for the purpose of increasing productionof isoprenoid compounds, and are not an exhaustive list of approachesthat can be taken. Numerous other strategies could be successfullyapplied to achieve this goal.

Blocking Pathways that Compete for FPP or GGPP.

In yeast, FPP is a branch point intermediate leading to the biosynthesisof sterols, heme, dolichol, ubiquinone, GGPP and farnesylated proteins.In E. coli, FPP serves as the substrate for octaprenyl pyrophosphatesynthase in the pathway leading to ubiquinone. In bacteria thatsynthesize carotenoids, such as Erwina uredovora, FPP is converted toGGPP by GGPP synthase in the first step leading to the carotenoids. Toincrease the production of farnesol or GG, it is desirable to inactivategenes encoding enzymes that use FPP or GGPP as substrate, or to reducethe activity of the enzymes themselves, either through mutation or theuse of specific enzyme inhibitors (as was discussed above for squalenesynthase). In S. cerevisiae, for example, it may be advantageous toinactivate the first step in the pathway from FPP to heme, in additionto inactivating ERG9. As discussed earlier, in E. coli, partial orcomplete inactivation of the octaprenyl pyrophosphate synthase couldincrease the availability of FPP for conversion of farnesol. Finally, inbacteria that produce carotenoids, such as Erwina uredovora, eliminationof GGPP synthase can increase the level of FPP for conversion offarnesol, while inactivating or reducing the activity of phytoenesynthase (the crtB gene product) can increase the level of GGPPavailable for conversion to GG.

It is possible that blocking pathways leading away from FPP or GGPPcould have negative effects on the growth and physiology of theproduction organism. It is further contemplated that additional geneticmodifications required to offset these complications can be made. Theisolation of mutants of S. cerevisiae that are blocked in the isoprenoidpathway and take up sterols under aerobic conditions, as describedabove, illustrates that compensating mutations can be obtained thatovercome the effects of the primary genetic modifications.

Isolation of Production Strains that are Resistant to Farnesol or GG.

In the Examples section, production of high levels of farnesol and GG bygenetically modified strains of S. cerevisiae is described. It isrecognized that as further increases in farnesol or GG production aremade, these compounds may reach levels that are toxic to the productionorganism. Indeed, product toxicity is a common problem encountered inbiological production processes. However, just as common are the geneticmodifications made by classical methods or recombinant technology thatovercome product toxicity. The present invention anticipatesencountering product toxicity. Thus a further embodiment of thisinvention is the isolation of mutants with increased resistance tofarnesol and/or GG.

Isolation of Production Organisms with Improved Growth Properties.

One effect of blocking the isoprenoid pathway in S. cerevisiae is thatthe mutant organisms (in the present invention, erg9 mutants) grow moreslowly than their parent (unblocked) strains, despite the addition ofergosterol to the culture medium. That the slower growth of the erg9mutants is due to the block at erg9 is illustrated in Example 1.G, whichshows that repairing the erg9 mutation restores the growth rate of thestrain to about that of the wild-type parent. The slower growth of theerg9 mutants could be due to differences related to growing onexogenously supplied ergosterol vs. ergosterol synthesized in the cell,or could be due to other physiological factors. One embodiment of thepresent invention is to isolate variants of the farnesol or GG producingstrains with improved growth properties. This could be achieved, forexample by continuous culture, selecting for faster growing variants.Such variants could occur spontaneously or could be obtained byclassical mutagenesis or molecular genetic approaches.

3.0 Incorporation of Prenol and Isoprenol

In a further embodiment of the present invention, farnesol or GG isproduced by the introduction of isoprenol and/or prenol into afermentation medium. With reference to FIGS. 7A-7B, each of thesecompounds, when taken up by an organism is phosphorylated with apyrophosphate group to form, respectively, 3-isopentenyl pyrophosphateand 3,3-dimethylallyl pyrophosphate. These two compounds interconvert bythe action of isopentenyl pyrophosphate isomerase. These compounds arethen converted to farnesyl pyrophosphate by the action of FPP synthase.Farnesol is formed by dephosphorylation of farnesyl pyrophosphate.Farnesyl pyrophosphate can be further converted to geranylgeranylpyrophosphate. Geranylgeraniol is formed be dephosyphorylation ofgeranylgeranyl pyrophosphate. GG would be formed from FPP by thecombined action of GGPP synthase and a phosphatase. Isoprenol and prenolare commercially available compounds and can be produced by methodsknown in the art

In this embodiment, the microorganism used in the fermentation can beany microorganism as described elsewhere herein. In addition, themicroorganism can be genetically modified to increase the action ofdimethylallyl transferase to promote the production of geranylpyrophosphate and farnesol pyrophosphate. Genes coding for this enzymefrom a variety of sources have been identified [Chen et al., ProteinSci., 3, 600-607 (1994)]. In addition a microorganism can be geneticallymodified to increase the action of isoprenol kinase or prenol kinase.Although these enzymes have not been discovered, similar enzymes thatphosphorylate farnesol and geranylgeraniol have been described[Bentinger et al., Arch. Biochem. Biophys., 353, 191-198 (1998); Ohnumaet al., J. Biochem., 119, 541-547 (1996)].

4.0 Fermentation Media and Conditions

In the method for production of farnesol or GG, a microorganism having agenetically modification, as discussed above is cultured in afermentation medium for production of farnesol or GG. An appropriate, oreffective, fermentation medium refers to any medium in which agenetically modified microorganism of the present invention, whencultured, is capable of producing farnesol or GG. Such a medium istypically an aqueous medium comprising assimilable carbon, nitrogen andphosphate sources. Such a medium can also include appropriate salts,minerals, metals and other nutrients. In addition, when an organismwhich is blocked in the ergosterol pathway and requires exogenoussterols, the fermentation medium must contain such exogenous sterols.Appropriate exemplary media are shown in the discussion below and in theExamples section. It should be recognized, however, that a variety offermentation conditions are suitable and can be selected by thoseskilled in the art.

Sources of assimilable carbon which can be used in a suitablefermentation medium include, but are not limited to, sugars and theirpolymers, including, dextrin, sucrose, maltose, lactose, glucose,fructose, mannose, sorbose, arabinose and xylose; fatty acids; organicacids such as acetate; primary alcohols such as ethanol and n-propanol;and polyalcohols such as glycerine. Preferred carbon sources in thepresent invention include monosaccharides, disaccharides, andtrisaccharides. The most preferred carbon source is glucose.

The concentration of a carbon source, such as glucose, in thefermentation medium should promote cell growth, but not be so high as torepress growth of the microorganism used. Typically, fermentations arerun with a carbon source, such as glucose, being added at levels toachieve the desired level of growth and biomass, but at undetectablelevels (with detection limits being about <0.1 g/l). In otherembodiments, the concentration of a carbon source, such as glucose, inthe fermentation medium is greater than about 1 g/L, preferably greaterthan about 2 g/L, and more preferably greater than about 5 g/L. Inaddition, the concentration of a carbon source, such as glucose, in thefermentation medium is typically less than about 100 g/L, preferablyless than about 50 g/L, and more preferably less than about 20 g/L. Itshould be noted that references to fermentation component concentrationscan refer to both initial and/or ongoing component concentrations. Insome cases, it may be desirable to allow the fermentation medium tobecome depleted of a carbon source during fermentation.

Sources of assimilable nitrogen which can be used in a suitablefermentation medium include, but are not limited to, simple nitrogensources, organic nitrogen sources and complex nitrogen sources. Suchnitrogen sources include anhydrous ammonia, ammonium salts andsubstances of animal, vegetable and/or microbial origin. Suitablenitrogen sources include, but are not limited to, protein hydrolysates,microbial biomass hydrolysates, peptone, yeast extract, ammoniumsulfate, urea, and amino acids. Typically, the concentration of thenitrogen sources, in the fermentation medium is greater than about 0.1g/L, preferably greater than about 0.25 g/L, and more preferably greaterthan about 1.0 g/L. Beyond certain concentrations, however, the additionof a nitrogen source to the fermentation medium is not advantageous forthe growth of the microorganisms. As a result, the concentration of thenitrogen sources, in the fermentation medium is less than about 20 g/L,preferably less than about 10 g/L and more preferably less than about 5g/L. Further, in some instances it may be desirable to allow thefermentation medium to become depleted of the nitrogen sources duringfermentation.

The effective fermentation medium can contain other compounds such asinorganic salts, vitamins, trace metals or growth promoters. Such othercompounds can also be present in carbon, nitrogen or mineral sources inthe effective medium or can be added specifically to the medium.

The fermentation medium can also contain a suitable phosphate source.Such phosphate sources include both inorganic and organic phosphatesources. Preferred phosphate sources include, but are not limited to,phosphate salts such as mono or dibasic sodium and potassium phosphates,ammonium phosphate and mixtures thereof. Typically, the concentration ofphosphate in the fermentation medium is greater than about 1.0 g/L,preferably greater than about 2.0 g/L and more preferably greater thanabout 5.0 g/L. Beyond certain concentrations, however, the addition ofphosphate to the fermentation medium is not advantageous for the growthof the microorganisms. Accordingly, the concentration of phosphate inthe fermentation medium is typically less than about 20 g/L, preferablyless than about 15 g/L and more preferably less than about 10 g/L.

A suitable fermentation medium can also include a source of magnesium,preferably in the form of a physiologically acceptable salt, such asmagnesium sulfate heptahydrate, although other magnesium sources inconcentrations which contribute similar amounts of magnesium can beused. Typically, the concentration of magnesium in the fermentationmedium is greater than about 0.5 g/L, preferably greater than about 1.0g/L, and more preferably greater than about 2.0 g/L. Beyond certainconcentrations, however, the addition of magnesium to the fermentationmedium is not advantageous for the growth of the microorganisms.Accordingly, the concentration of magnesium in the fermentation mediumis typically less than about 10 g/L, preferably less than about 5 g/L,and more preferably less than about 3 g/L. Further, in some instances itmay be desirable to allow the fermentation medium to become depleted ofa magnesium source during fermentation.

The fermentation medium can also include a biologically acceptablechelating agent, such as the dihydrate of trisodium citrate. In suchinstance, the concentration of a chelating agent in the fermentationmedium is greater than about 0.2 g/L, preferably greater than about 0.5g/L, and more preferably greater than about 1 g/L. Beyond certainconcentrations, however, the addition of a chelating agent to thefermentation medium is not advantageous for the growth of themicroorganisms. Accordingly, the concentration of a chelating agent inthe fermentation medium is typically less than about 10 g/L, preferablyless than about 5 g/L, and more preferably less than about 2 g/L.

The fermentation medium can also initially include a biologicallyacceptable acid or base to maintain the desired pH of the fermentationmedium. Biologically acceptable acids include, but are not limited to,hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid andmixtures thereof. Biologically acceptable bases include, but are notlimited to, ammonium hydroxide, sodium hydroxide, potassium hydroxideand mixtures thereof. In a preferred embodiment of the presentinvention, the base used is ammonium hydroxide.

The fermentation medium can also include a biologically acceptablecalcium source, including, but not limited to, calcium chloride.Typically, the concentration of the calcium source, such as calciumchloride, dihydrate, in the fermentation medium is within the range offrom about 5 mg/L to about 2000 mg/L, preferably within the range offrom about 20 mg/L to about 1000 mg/L, and more preferably in the rangeof from about 50 mg/L to about 500 mg/L.

The fermentation medium can also include sodium chloride. Typically, theconcentration of sodium chloride in the fermentation medium is withinthe range of from about 0.1 g/L to about 5 g/L, preferably within therange of from about 1 g/L to about 4 g/L, and more preferably in therange of from about 2 g/L to about 4 g/L.

As previously discussed, the fermentation medium can also include tracemetals. Such trace metals can be added to the fermentation medium as astock solution that, for convenience, can be prepared separately fromthe rest of the fermentation medium. A suitable trace metals stocksolution for use in the fermentation medium is shown below in Table 1.Typically, the amount of such a trace metals solution added to thefermentation medium is greater than about 1 ml/L, preferably greaterthan about 5 mL/L, and more preferably greater than about 10 mL/L Beyondcertain concentrations, however, the addition of a trace metals to thefermentation medium is not advantageous for the growth of themicroorganisms. Accordingly, the amount of such a trace metals solutionadded to the fermentation medium is typically less than about 100 mL/L,preferably less than about 50 mL/L, and more preferably less than about30 mL/L It should be noted that, in addition to adding trace metals in astock solution, the individual components can be added separately, eachwithin ranges corresponding independently to the amounts of thecomponents dictated by the above ranges of the trace metals solution.

As shown below in Table 1, a suitable trace metals solution for use inthe present invention can include, but is not limited to ferroussulfate, heptahydrate; cupric sulfate, pentahydrate; zinc sulfate,heptahydrate; sodium molybdate, dihydrate; cobaltous chloride,hexahydrate; and manganous sulfate, monohydrate. Hydrochloric acid isadded to the stock solution to keep the trace metal salts in solution.

TABLE 1 TRACE METALS STOCK SOLUTION CONCENTRATION COMPOUND (mg/L)Ferrous sulfate heptahydrate 280 Cupric sulfate, pentahydrate 80 Zinc(II) sulfate, heptahydrate 290 Sodium molybdate, dihydrate 240 Cobaltouschloride, hexahydrate 240 Manganous sulfate, monohydrate 170Hydrochloric acid 0.1 ml

The fermentation medium can also include vitamins. Such vitamins can beadded to the fermentation medium as a stock solution that, forconvenience, can be prepared separately from the rest of thefermentation medium. A suitable vitamin stock solution for use in thefermentation medium is shown below in Table 2. Typically, the amount ofsuch vitamin solution added to the fermentation medium is greater than 1ml/L, preferably greater than 5 ml/L and more preferably greater than 10ml/L. Beyond certain concentrations, however, the addition of vitaminsto the fermentation medium is not advantageous for the growth of themicroorganisms. Accordingly, the amount of such a vitamin solution addedto the fermentation medium is typically less than about 50 ml/L,preferably less than 30 ml/L and more preferably less than 20 ml/L. Itshould be noted that, in addition to adding vitamins in a stocksolution, the individual components can be added separately each withinthe ranges corresponding independently to the amounts of the componentsdictated by the above ranges of the vitamin stock solution.

As shown in Table 2, a suitable vitamin solution for use in the presentinvention can include, but is not limited to, biotin, calciumpantothenate, inositol, pyridoxine-HCl and thiamine-HCl.

TABLE 2 COMPOUND CONCENTRATION (mg/L) Biotin 10 Calcium pantothenate 120Inositol 600 Pyridoxine-HCl 120 Thiamine-HCl 120

As stated above, when an organism is blocked in the sterol pathway, anexogenous sterol must be added to the fermentation medium. Such sterolsinclude, but are not limited to, ergosterol and cholesterol. Suchsterols can be added to the fermentation medium as a stock solution thatis prepared separately from the rest of the fermentation medium. Sterolstock solutions can be prepared using a detergent to aid insolubilization of the sterol. A typical ergosterol stock solution isdescribed in Example 1.D. Typically, an amount of sterol stock solutionis added to the fermentation medium such that the final concentration ofthe sterol in the fermentation medium is within the range of from about1 mg/L to 3000 mg/L, preferably within the range from about 2 mg/L to2000 mg/L, and more preferably within the range from about 5 mg/L to2000 mg/L.

Microorganisms of the present invention can be cultured in conventionalfermentation modes, which include, but are not limited to, batch,fed-batch, cell recycle, and continuous. It is preferred, however, thatthe fermentation be carried out in fed-batch mode. In such a case,during fermentation some of the components of the medium are depleted.It is possible to initiate the fermentation with relatively highconcentrations of such components so that growth is supported for aperiod of time before additions are required. The preferred ranges ofthese components are maintained throughout the fermentation by makingadditions as levels are depleted by fermentation. Levels of componentsin the fermentation medium can be monitored by, for example, samplingthe fermentation medium periodically and assaying for concentrations.Alternatively, once a standard fermentation procedure is developed,additions can be made at timed intervals corresponding to known levelsat particular times throughout the fermentation. As will be recognizedby those in the art, the rate of consumption of nutrient increasesduring fermentation as the cell density of the medium increases.Moreover, to avoid introduction of foreign microorganisms into thefermentation medium, addition is performed using aseptic additionmethods, as are known in the art. In addition, a small amount ofanti-foaming agent may be added during the fermentation.

The temperature of the fermentation medium can be any temperaturesuitable for growth and production of farnesol or GG. For example, priorto inoculation of the fermentation medium with an inoculum, thefermentation medium can be brought to and maintained at a temperature inthe range of from about 20° C. to about 45° C., preferably to atemperature in the range of from about 25° C. to about 40° C., and morepreferably in the range of from about 28° C. to about 32° C.

The pH of the fermentation medium can be controlled by the addition ofacid or base to the fermentation medium. In such cases when ammonia isused to control pH, it also conveniently serves as a nitrogen source inthe fermentation medium. Preferably, the pH is maintained from about 3.0to about 8.0, more preferably from about 3.5 to about 7.0, and mostpreferably from about 4.0 to about 6.5.

The fermentation medium can also be maintained to have a dissolvedoxygen content during the course of fermentation to maintain cell growthand to maintain cell metabolism for production of farnesol or GG. Theoxygen concentration of the fermentation medium can be monitored usingknown methods, such as through the use of an oxygen electrode. Oxygencan be added to the fermentation medium using methods known in the art,for, through agitation and aeration of the medium by stirring, shakingor sparging. Preferably, the oxygen concentration in the fermentationmedium is in the range of from about 20% to about 100% of the saturationvalue of oxygen in the medium based upon the solubility of oxygen in thefermentation medium at atmospheric pressure and at a temperature in therange of from about 20° C. to about 40° C. Periodic drops in the oxygenconcentration below this range may occur during fermentation, however,without adversely affecting the fermentation.

Although aeration of the medium has been described herein in relation tothe use of air, other sources of oxygen can be used. Particularly usefulis the use of an aerating gas which contains a volume fraction of oxygengreater than the volume fraction of oxygen in ambient air. In addition,such aerating gases can include other gases which do not negativelyaffect the fermentation.

In an embodiment of the fermentation process of the present invention, afermentation medium is prepared as described above and in Example 1.H.This fermentation medium is inoculated with an actively growing cultureof microorganisms of the present invention in an amount sufficient toproduce, after a reasonable growth period, a high cell density. Typicalinoculation cell densities are within the range of from about 0.01 g/Lto about 10 g/L, preferably from about 0.2 g/L to about 5 g/L and morepreferably from about 0.05 g/L to about 1.0 g/L, based on the dry weightof the cells. In production scale fermentors, however, greater inoculumcell densities are preferred. The cells are then grown to a cell densityin the range of from about 10 g/L to about 100 g/L preferably from about20 g/L to about 80 g/L, and more preferably from about 50 g/L to about70 g/L. The residence times for the microorganisms to reach the desiredcell densities during fermentation are typically less than about 200hours, preferably less than about 120 hours, and more preferably lessthan about 96 hours.

In one mode of operation of the present invention, the carbon sourceconcentration, such as the glucose concentration, of the fermentationmedium is monitored during fermentation. Glucose concentration of thefermentation medium can be monitored using known techniques, such as,for example, use of the glucose oxidase enzyme test or high pressureliquid chromatography, which can be used to monitor glucoseconcentration in the supernatant, e.g., a cell-free component of thefermentation medium. As stated previously, the carbon sourceconcentration should be kept below the level at which cell growthinhibition occurs. Although such concentration may vary from organism toorganism, for glucose as a carbon source, cell growth inhibition occursat glucose concentrations greater than at about 60 g/L, and can bedetermined readily by trial. Accordingly, when glucose is used as acarbon source the glucose is preferably fed to the fermentor andmaintained below detection limits. Alternatively, the glucoseconcentration in the fermentation medium is maintained in the range offrom about 1 g/L to about 100 g/L, more preferably in the range of fromabout 2 g/L to about 50 g/L, and yet more preferably in the range offrom about 5 g/L to about 20 g/L. Although the carbon sourceconcentration can be maintained within desired levels by addition of,for example, a substantially pure glucose solution, it is acceptable,and may be preferred, to maintain the carbon source concentration of thefermentation medium by addition of aliquots of the original fermentationmedium. The use of aliquots of the original fermentation medium may bedesirable because the concentrations of other nutrients in the medium(e.g. the nitrogen and phosphate sources) can be maintainedsimultaneously. Likewise, the trace metals concentrations can bemaintained in the fermentation medium by addition of aliquots of thetrace metals solution.

5.0 Farnesol and GG Recovery

Once farnesol or GG are produced by a biological system, they arerecovered or isolated for subsequent use. The present inventors haveshown that for both farnesol and GG, the product may be present inculture supernatants and/or associated with the yeast cells. Withrespect to the cells, the recovery of farnesol or GG includes somemethod of permeabilizing or lysing the cells. The farnesol or GG in theculture can be recovered using a recovery process including, but notlimited to, chromatography, extraction, solvent extraction, membraneseparation, electrodialysis, reverse osmosis, distillation, chemicalderivatization and crystallization. When the product is in the phosphateform, i.e., farnesyl phosphate or geranylgeranyl phosphate, it onlyoccurs inside of cells and therefore, requires some method ofpermeabilizing or lysing the cells.

The following Examples are provided to illustrate embodiments of thepresent invention and are not intended to limit the scope of theinvention as set forth in the claims.

EXAMPLES Example 1

This example describes the creation of erg9 mutants by chemicalmutagenesis of S. cerevisiae strain ATCC 28383 using nitrous acid.

As noted above, one way of increasing yields of farnesol or GG is todecrease or eliminate squalene synthase activity. In S. cerevisiae,squalene synthase is encoded by the ERG9 gene.

A. Mutagenesis

Strain 28383 was obtained from the ATCC. It is the parent of strain60431 used below in some experiments for comparative purposes (alsoobtained from ATCC). Strain 60431 is an erg9-1 mutant that producesfarnesol.

A kill curve was performed using nitrous acid with ATCC 28383. Usinginformation derived from the kill curve, a mutagenesis of ATCC 28383with nitrous acid was performed as follows. The 28383 cells wereincubated overnight at 30° C. in YPD medium (1% Bacto-yeast extract, 2%Bacto-peptone, and 2% glucose) with shaking. The cells were washed andmutagenized. After mutagenesis, the cells were allowed to recover byculturing them in YPDC (YPD plus 4 mg/L cholesterol) at 22° C.overnight. The culture was then washed and plated onto YPDC agar (YPDCplus 2% Bacto-agar) containing various levels of nystatin (20, 30, 40,50 mg/L). These cultures were incubated for two weeks at 22° C. Theantifungal polyene antibiotic nystatin inhibits cell wall synthesis ingrowing cells by binding specifically to ergosterol, which, in turn,results in an alteration of selective permeability. Some strains thatare resistant to nystatin have been shown to have decreased productionof ergosterol. Nystatin has little, if any, affinity for cholesterolwhich, when supplied exogenously, can be used by sterol auxotrophs asefficiently as ergosterol.

Approximately 3,000 nystatin-resistant colonies were obtained. They werescreened for temperature sensitivity (ts) and sterol auxotrophy. Todetermine temperature sensitivity, mutagenized cells were grown on YPDCat a permissive temperature (22° C.) and subsequently replica plated toYPD agar and incubated at a restrictive temperature (37° C.). Any cellswhich fail to grow at the higher temperature would include those withboth a conditionally lethal and a constitutively lethal defect in thesterol pathway. Temperature sensitive mutants were screened for sterolauxotrophy by replica plating to YPD agar (lacking cholesterol) andincubation overnight at 28° C. Of the 3,000 nystatin-resistant colonies,170 were saved after confirmation of ts and sterol auxotrophy.

B. Tube Assays

The 170 strains were initially evaluated in tube assays for theproduction of farnesol and other intermediates of the isoprenoid pathwayas follows. Cells were cultured in 10 ml YPDC in tubes with screw capsfor 48 hours at 28° C. with shaking. The cells were centrifuged for 5-10minutes at 2800 rpm, and the supernatant was transferred to anothertube. The cells were washed once with 5 ml distilled water andcentrifuged again for 5-10 minutes at 2800 rpm.

The cell pellet was extracted by adding 2.5 ml 0.2% pyrogallol (inmethanol) and 1.25 ml 60% KOH (in distilled H₂O), vortexing to mix, andincubating in a 70-75° C. water bath for 1.5 hours. After thissaponification, 5 ml hexane were added, and the tube was recapped andvortexed for 15-30 seconds. The tube was centrifuged at 2800 rpm forminutes to separate the phases. The hexane layer was recovered. Ifnecessary, the sample can be concentrated by evaporating solvent undernitrogen and adding back an appropriate amount of hexane for analysis.

To extract the supernatant, 5 ml of hexane were added to thesupernatant, and the tube was recapped and vortexed for 15-30 seconds.The tube was centrifuged at 2800 rpm for 25 minutes to separate phases.The hexane layer was recovered, and the sample was concentrated byevaporating under nitrogen and adding back an appropriate amount ofhexane for analysis.

The hexane extracts from this primary screen were analyzed by thin layerchromatography (TLC) with confirmation by gas chromatography (GC)/massspectrometry (MS) for the presence of farnesol and other intermediatesof the isoprenoid pathway. TLC was performed on reversed-phase C18silica gel plates using 6:4 (v/v) ethylacetate/acetonitrile (EtOAc/MeCN)as the mobile phase and 2% (w/v) phosphomolybdic acid (PMA) in ethanolfor detection. GC/MS was performed using a Hewlett Packard 5890 GC and a5970 series mass selective detector. The column used was a RestekRtx-5MS (15 m length, 0.25 mm ID, 1 μm film system. Thirteen mutantswere identified as producers of farnesol or other intermediates.

C. Shake Flask Assays

These thirteen mutants were then screened in shake flasks, along withthe parental strain, 28383, and the farnesol producing strain, 64031.Fifty ml of YPDC medium in 250 ml baffled Erlenmeyer flasks wereinoculated with overnight cultures to an initial OD at 600 nm of 0.05and incubated with shaking at 28° C. The flasks were sampled, and OD at600 nm, dry weight and sterol content were determined. To measure dryweight, 5 ml of the culture were centrifuged at 8,000 rpm. The cellswere washed twice with distilled H₂O, and the cell pellet wasresuspended in 3-5 ml distilled H₂O and transferred to a pre-weighed dryweight pan. The pan was placed in 100° C. oven for 24 hours, then cooledin a desiccator, weighed on an analytical balance, and the dry weight ing/L determined from the formula:

[Total weight (pan+cells)−pan weight]×200=dry weight in g/L.

For sterol determination, 20 ml of culture were processed, the cellpellet and supernatant extracted, and farnesol and other sterolsmeasured as described above.

Seven of the 13 mutants appeared to produce intermediates in theergosterol pathway and were evaluated further in other shake flaskexperiments (see below). All 13 mutants had been selected by resistanceto the highest levels of nystatin.

Overnight cultures were used to inoculate 50 ml of YPDC medium intriplicate shake flasks for each of the seven mutant strains (MBNA1-1,MBNA1-5, MBNA1-9, MBNA1-10, MBNA1-11, MBNA1-13, MBNA1-14,), the parentalstrain 28383 and mutant strain 64031. The cells and medium wereextracted separately at 24, 48, and 72 hours into hexane, as describedabove. Dry weight analysis (performed as described above) was performedat each of these time points to determine cell density. The hexaneextracts were analyzed by GC/MS for their farnesol, squalene,cholesterol, and ergosterol levels. The farnesol results are presentedin Table 3 below.

Low levels of farnesol were found in the culture supernatants with muchhigher levels accumulating in the cells. Strain MBNA1-1 produced 1%farnesol and also made 0.1% ergosterol at 72 hours. Strain MBNA1-5 didnot make any farnesol, but produced 0.3% squalene. Strain MBNA1-9produced 0.64% farnesol in 48 hours. MBNA1-10, MBNA1-11, and MBNA1-14produced very low levels of farnesol in the cells and appeared to makeergosterol. MBNA1-13 showed the highest farnesol production (2.5%) basedon cell dry weight (42,187 ng/ml culture) and did not show anyergosterol production. Thus, among the farnesol-producing strains(MBNA1-1, MBNA1-9 and MBNA1-13), there appeared to be different degreesof blockage in the ergosterol pathway, since MBNA1-1 and MBNA1-9produced lower levels of farnesol and did not require ergosterol forgrowth, and MBNA1-13 produced the highest level of farnesol and had astrict requirement for sterol supplementation for growth. The parentalstrain 28383 showed ergosterol production up to 0.4% (no farnesolproduction), and erg9 mutant strain 64031 showed farnesol accumulationto approximately 0.4%.

TABLE 3 Characterization of New Mutants in Shake Flask Experiment Hoursof Dry weight Farnesol (ng/ml Farnesol (% of Strain Name Growth mg/ml ofculture) cell dry weight) MBNA1-1 24 0.16 0 0.00 48 1.12 1793 0.16 721.26 13187 1.05 MBNA1-5 24 2.46 0 0.00 48 1.6 0 0.00 72 1.06 0 0.00MBNA1-9 24 1.32 1122 0.09 48 2.96 19062 0.64 72 4.34 70625 0.00 MBNA1-1024 4.76 4636 0.10 48 9.44 444 0.00 72 7.9 166 0.00 MBNA1-11 24 1.82 00.00 48 3.58 258 0.01 72 2.36 281 0.01 MBNA1-13 24 0.18 0 0.00 48 3.123657 0.12 72 1.66 42187 2.54 MBNA1-14 24 2.42 0 0.00 48 8.98 0 0.00 727.36 0 0.00 ATCC28383 24 7.36 0 0.00 48 8.52 0 0.00 72 8.84 0 0.00ATCC64031 24 1.58 90 0.01 48 1.84 6845 0.37 72 1.28 4874 0.38

D. Enzyme Analysis

For all enzyme assays, cells were grown in YPDE medium. (YPD mediumcontaining 5 mg/L ergosterol). The cells were harvested bycentrifugation. One gram of cells (wet weight) was suspended in 4 ml of0.1 M Tris HCl, pH 7.0. The cell suspension was then disrupted bypassage through a French pressure cell (20,000 psi). The resulting(lysed) cell suspension was then centrifuged (15,000×g) and thesupernatant (cell free extract) was used directly for enzyme assaysunless noted otherwise.

The mutants were assayed for squalene synthase activity. The squalenesynthase assay involved incubation of a cell-free extract with FPP inthe presence the reduced form of nicotinamide adenine dinucleotidephosphate, (NADPH), extraction into ethyl acetate, and squalenedetection by GC. Specifically, 0.1 M Tris/HCl pH7 (X μL), 0.1 M DTT (2μL), 0.1 M MgCl₂ (10 μL), 0.1 M NADPH (4 μL), 1 mg/mL FPP (6 μL), andcell-free extract (7000×g supernatant) (Y μL), where X+Y=178 μL to make200 μL total, were combined. This mixture was incubated in glass tubesat 37° C. for 40 min. Then, it was extracted with 0.15 mL ethyl acetate.The extract was transferred to plastic vials and centrifuged at 15,000×gfor 5 min. The ethyl acetate extract was analyzed using GC/MS.

Table 4 shows squalene synthase levels for mutants MBNA1-1, MBNA1-9,MBNA1-13, and ATCC strains 64031 and 28383. Squalene synthase activitywas 0.12 μg squalene formed/min×mg protein for the wild-type organism28383. The activity levels for the other four strains were less than thedetectable limits of this assay. Reduced squalene synthase levels wouldbe expected for MBNA1-1, MBNA1-9 and 64031 since they have beencharacterized as being partially-blocked mutants that produce farnesoland low amounts of ergosterol. MBNA1-13 is a farnesol-producing mutantwhich cannot grow without added sterol, so this blocked mutant would beexpected to have no detectable level of squalene synthase. The extractsof these strains were reassayed with higher concentrations of cell-freeextract and longer incubation times. Once again, squalene synthase wasnot detected in any of the mutants.

TABLE 4 Squalene Synthase Assay Strain (μg/min × mg protein) MBNA1-1 NDMBNA1-9 ND MBNA1-13 ND ATCC 64031 ND ATCC 28383 0.12 ND = Not detected

An enzyme analysis was performed for the farnesol-producing strains(MBNA1-1, MBNA1-9 and MBNA1-13) and their parental strain, 28283. Theenzyme levels compared were those of the acetoacetyl CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and a phosphatase.Cell-free extracts were prepared as described above.

For the FPP synthase assay, 0.1 M DTT (2 μL), 0.1 M MgCl₂ (2 μL), 1mg/mL IPP (6 μL), 1 mg/mL GPP (6 μL), cell-free extract (Y), and 0.1 MTris/HCl (pH 7.0) (X μL), where X+Y=84 μL, were combined, and incubatedat 37° C. for 15 min. This mixture was then extracted twice with 0.3 mLhexane to remove pre-existing farnesol. Next, 0.1 mL of 2× glycinebuffer (0.2 M glycine, 2 mM MgCl₂, 2 mM ZnCl₂), pH 10.4, and 33 units ofalkaline phosphatase were added, and the mixture was incubated at 37° C.for an hour. The mixture was extracted with 0.1 mL of ethyl acetate anddried with sodium sulfate. Farnesol was determined with GC. Ano-phosphatase control was included in each assay.

HMG-CoA reductase catalyzes the reaction of HMG-CoA with NADPH to formmevalonate, nicotinamide adenine dinucleotide phosphate (NADP) and CoA(see FIG. 1). For these assays, the cell-free extracts were prepared asdescribed above except that the disrupted cell suspensions werecentrifuged at 7,000×g instead of 15,000×g. The reaction was followed bymonitoring consumption of HMG-CoA by high performance liquidchromatography (HPLC). For the assay, a reaction mixture containing (ina final volume of 0.1 ml) 0.1 M Na₂H₂P₂O₇, pH 6.5, 2 mM DTT, 1 mM NADPH,0.4 mM HMG-CoA, and cell-free extract (the volume of extract was varied,with the balance of the 0.1 ml reaction made up with 0.1 M Na₂H₂P₂O₇, pH6.5) was incubated at 37° C. for 15 minutes. The reaction was monitoredwith HPLC (Luna C18 (Phenomenex) reversed-phase column using a gradientof 0% Solvent B for 1 min. followed by 0-11% B over 42 min. Solvent Awas 86:14 (v/v) 40 mM potassium phosphate (pH 6.0)/methanol. Solvent Bwas methanol. The wavelength for detection was 260 nm).

HMG-CoA synthase converts acetoacetyl CoA and acetyl CoA to HMG-CoA andCoA. The assay monitors the production of HMG-CoA by HPLC. For theassay, 0.1 M Na₂H₂P₂O₇, pH 6.5, (X μL), 20 mM acetoacetyl CoA (4 μL), 20mM acetyl CoA (2 μL), and cell-free extract (Y μL), where X+Y is 94 μL(total volume 100 μL), were combined and incubated at 37° C. for 5minutes. Then, the reaction mixture was heated at 60° C. for 5 minutesto inactive the enzyme, followed by centrifugation in a microfuge atfull speed for 5 minutes. The supernatant was analyzed by HPLC.

Acetoactyl CoA thiolase catalyzes a reversible reaction whereby, in theforward direction, two molecules of acetyl CoA are reacted to formacetoacetyl CoA and CoA. The assay monitors the formation of acetyl CoA(reverse reaction) by HPLC. For the assay, 0.1 M Na₂H₂P₂O₇, pH 6.5, (XμL), 20 mM acetoacetyl CoA (2 μL), 20 mM CoA (3 μL), and cell-freeextract (Y μL), where X+Y is 95 μL (total volume 100 μL), were combinedand incubated at 37° C. for 5 minutes. Then, the reaction mixture wascentrifuged in a Microcon-10 (Amicon) centrifuge to separate the enzymefrom the product. The supernatant was analyzed by HPLC.

The phosphatase assay quantitates phosphatase activity by measuring theformation of p-nitrophenol (increased absorbance at 400 nm). For thephosphatase assay, 0.1 M Tris-HCl, pH 7 (X μl), 1 M MgCl₂ (2 μl), 50 mMp-nitrophenyl phosphate (10 μl) and cell-free extract (Y μl), whereX+Y=1 mL, were combined and incubated at 37° C. for 15 minutes, afterwhich the absorbance at 400 nm was measured.

Table 5 shows the levels for each of the five enzymes in these fourstrains. The acetoacetyl CoA thiolase levels were comparable in all ofthese strains. The HMG-CoA synthase and HMG-CoA reductase levelsappeared to be two to three times higher in the mutant strains. The FPPsynthase levels were the same or lower in the mutants than in 28383. Thephosphatase levels appeared to be elevated three to four fold in themutant strains compared to the parent. Since all of these strains wereisolated by classical mutagenesis, it is possible that other mutationsexist in these strains, besides the erg9 mutation.

TABLE 5 ATCC MBNA1-1 MBNA1-9 MBNA1-13 28383 Acetoacetyl CoA 2.4 ×. 10²2.7 ×. 10² 1.6 × 10² 2.0 × 10² Thiolase (nmol AcCoA/min × mg protein)HMG-CoA Synthase 5.7 7.2 7.1 2.7 (nmol HMG-CoA/ min × mg protein)HMG-CoA Reductase 0.35 0.55 0.36 0.15 (nmol CoA/min × mg protein) FPPSynthase 1.3 2.0 1.6 2.2 (nmol Farnesol/min × mg protein) GeneralPhosphatase 82 83 91 24 (nmol p-nitrophenol/ min × mg protein)

E. ERG9 Genetic Analysis

Chromosomal DNA was prepared from strains ATCC 28383, MBNA1-1, MBNA1-9and MBNA1-13 according to the method described in Sherman et al.(Sherman, F., G. R. Fink, and J. B. Hicks, 1989, Methods in YeastGenetics, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.).The genomic DNA was digested with the restriction enzymes ApaI and NsiI,and subjected to Southern blot analysis using a biotinylated ERG9 DNAfragment as a probe. The ERG9 probe consisted of a 2044 bp ERG9 DNAfragment isolated from an agarose gel after digestion of the plasmidpTWM103 with ApaI and NsiI. pTWM103 consists of the ERG9 gene andflanking sequences extending from position #10916 to #13868 of GenBankAccession #U00030 sequence. This plasmid was constructed by polymerasechain reaction (PCR) amplification of the ERG9 gene and flanking regionsusing the following two oligonucleotides:

5′-oligo = VE107-5 (denoted herein as SEQ ID NO: 1)CTC AGT ACG CTG GTA CCC GTC AC 3′-oligo = VE105-3(denoted herein as SEQ ID NO: 2) gat gga TCC CAA TAT GTG TAG CTC AGG

Capital letters designate DNA sequences from ERG9 flanking region, whilesmall letters designate bases added to create restriction sites. VE107-5contains sequences from position #10905 to position #10928 of GenBankAccession #U00030 sequence. VE105-3 contains the reverse complement ofsequences from position #13868 to position #13847.

The amplification conditions were as follows:

Template DNA was genomic DNA isolated from strain S288C (Yeast GeneticStock Center, Berkeley, Calif.)2 min. 94° C./1 cycle1 min. 94° C., 1 min. 52° C., 1.5 min 74° C./30 cycles5 min. At 74° C./1 cycle

The 2969 bp ERG9 DNA generated from this PCR reaction was digested withKpnI and BamHI, then ligated to KpnI, BamHI digested YEp352 to generateplasmid pTWM103. YEp352 is a yeast/E. coli shuttle vector (Hill, J. E.,Myers, A. M., Koerner, T. J., and Tzagaloff, A., 1986, Yeast/E. coliShuttle Vectors with Multiple Unique Restriction Sites, Yeast 2:163-167).

DNA for the ERG9 probe described above was obtained by digesting TWM103with ApaI and NsiI, and purifying the 2044 bp fragment containing theERG9 gene and flanking sequences. The ERG9 DNA was then biotinylatedusing random primer extension (NEBlot Phototope Kit, New EnglandBioLabs, Beverly, Mass.).

Approximately 1 μg of biotinylated ERG9 probe was hybridized to theSouthern blot of ApaI, NsiI digested genomic DNA from strains ATCC28383, MBNA1-1, MBNA1-9, and MBNA1-13. The blot revealed that all fourstrains contained a single hybridizing sequence located on a 2044 bpfragment.

To clone the ERG9 genes from these four strains, genomic DNA from eachwas again digested with ApaI and NsiI. The yeast/E. coli shuttle vectorpRS315 (Sikorski, R. S., and P. Hieter, 1989, A System of ShuttleVectors and Yeast Host Strains Designed for Efficient Manipulation ofDNA in Saccharomyces cerevisiae, Genetics, 122: 19-27) was digested withApaI and PstI (NsiI and PstI have compatible cohesive ends). Thedigested DNAs were separated on an agarose gel, and chromosomal DNAfragments in the size range from approximately 1.6 kb to approximately 3kb, as well as the 3895 bp band corresponding to digested pRS316 werecut from the gel and purified using GeneClean (BIO 101, Inc., Visa,Calif.). The purified genomic DNA fragments from each of the fourstrains was ligated to pRS316, and transformed into E. coli.Transformants containing the pRS316/ERG9 clones were identified bycolony hybridization using the ERG9 probe described above. ERG9 clonesderived from each of the four strains were isolated in this manner. Thecloned ERG9 genes from each strain were sequenced by ACGT, Inc.,Northbrook, Ill., using an Applied Biosystems, Inc. automated DNAsequencer, Model ABI 100.

The sequence of the ERG9 gene from ATCC 28383 was identical to thesequence deposited in GenBank (Accession #U00030). The erg9 genes fromMBNA1-2 and MBNA1-9 were found to have the same mutation, namely, achange from G to A at position #12386 of GenBank Accession #U00030,which changes a TGG codon to a TGA stop codon. This causes terminationof the ERG9 protein at 237 amino acids instead of the normal 444 aminoacids found in the wild-type ERG9 protein.

The erg9 gene from MBNA1-13 was found to contain a deletion of a C atposition #12017 of GenBank Accession #U00030, which causes a frameshiftand early termination of the ERG9 protein at 116 amino acids, with thelast two amino acids being different from the wild-type ERG9 protein(due to the frameshift). The erg9 alleles from MBNA1-1 and MBNA1-9retain low levels of activity. This was determined by transforming anergosterol-requiring erg9 deletion mutant with plasmids that carry themutant erg9 genes from MBNA1-1 and MBNA1-9. The presence of the clonedgenes in the erg9 deletion strains allowed these transformants to grow,albeit slowly, on medium lacking ergosterol. The erg9 allele fromMBNA1-13 did not show any residual activity when tested in this manner.The erg9 deletion strain grew at wild-type levels when transformed withthe ERG9 allele from ATCC 28383.

F. Description of Secondary Mutations

Saccharomyces cerevisiae does not normally import ergosterol from itsenvironment under aerobic conditions. However, the isolated erg9 mutantswere able to grow aerobically when supplemented with ergosterol orcholesterol, and therefore, must contain secondary mutations that allowaerobic sterol uptake to occur. The methods used to isolate ergosterolpathway mutants, such as the erg9 mutant MBNA1-13, included not only aselection for ergosterol pathway mutants, but a selection for cells thatimport sterols under aerobic conditions as well, since only mutants withmutations in both a sterol pathway gene and a sterol uptake gene wouldhave survived.

In Example 3 below, the difficulty encountered when attempting to obtainerg9 knockout mutations by molecular methods, even in a strain carryingthe upc2 mutation, which is reported to allow uptake of sterols underaerobic conditions (Lewis, T. L., G. A. Keesler, G. P. Fenner and L. W.Parks, 1988, Pleiotropic mutations in Saccharomyces cerevisiae affectingsterol uptake and metabolism, Yeast 4:93-106) is described. It appearsthat the upc2 mutation does not confer efficient aerobic sterol uptakein an erg9 mutant, and that the strains described in Example 3 acquiredadditional spontaneous mutations (at a low frequency rate) that improvetheir ability to take up sterols aerobically.

Having the appropriate mutations that enhance aerobic uptake ofergosterol and cholesterol in an otherwise wild type strain allow one toisolate erg9 mutations in that strain at a much higher frequency thanwould be obtained with a non-sterol uptake mutant strain. In order todemonstrate that the erg9 mutant strain MBNA1-13 (and derivatives ofthis strain) had acquired a mutation(s) that enhances uptake of sterolsunder aerobic conditions (referred to as sterol uptake enhancement orsue mutations), the frequency of creating an erg9 knockout in theMBNA1-13 background, which contains the sterol uptake mutation, to thefrequency of obtaining the erg9 knockout mutation in the ATCC 28383parental background, which presumably lacks the sterol uptake mutation,was compared. This was done with the following two strains representingeach strain background. SWE23-ΔHE9 (ura3, his3, sue) was derived fromMBNA1-13 and contains a repaired ERG9 gene. SWY5-ΔH1 (ura3, his3) is anauxotrophic mutant derived from strain ATCC 28383. The construction ofSWE23-ΔHE9 and SWY5-ΔH1 is described in detail in Section G below. SinceSWE29-ΔHE9 was derived from MBNA1-13, this strain served as the hostcarrying the sue mutation, whereas SWY5-ΔH, which was derived from ATCC28383, served as the host without the sue mutation. Both strains weretransformed with equal amounts (approximately 1 μg) of the erg9Δ::HIS3DNA fragment obtained by digesting pKML19-41 (plasmid constructiondescribed in Example 3) with BamHI and XmaI, and purifying the resulting2.1 kb fragment. Cells were transformed with this 2.1 kb fragment usingthe LiAc transformation procedure (Gietz, R. D., R. H. Schiestl, A. R.Willems and R. A. Woods, 1995, Studies on the transformation of intactyeast cells by the LiAc/SS-DNA/PEG procedure, Yeast 11: 355-360), andtransformants were selected on SCE-ura medium. SCE medium contains 0.67%Bacto yeast nitrogen base (without amino acids), 2% dextrose, 20 mg/Ladenine sulfate, 20 mg/L uracil, 20 mg/L L-tryptophan, 20 mg/LL-histidine, 20 mg/L L-arginine, 20 mg/L L-methionine, 30 mg/LL-tyrosine, 30 mg/L L-leucine, 30 mg/L L-isoleucine, 30 mg/L L-lysine,50 mg/L L-phenylalanine, 100 mg/L L-glutamic acid, 100 mg/L L-asparticacid, 150 mg/L L-valine, 200 mg/L L-threonine, 400 mg/L L-serine and 5mg/L ergosterol. For agar plates, 2% Bacto agar is added to the SCEmedium. SCE-ura medium is SCE lacking uracil.

A total of 24 HIS⁺ colonies arose from the transformation of SWY5-ΔH1,while 401 HIS⁺ colonies were obtained from SWE23-ΔHE9. Since it waspossible for the erg9Δ::HIS3 gene to integrate at loci other than theERG9 locus (and therefore allowing the cells to remain prototrophic forergosterol), twenty of each type of transformant were tested for anergosterol requirement by plating onto YPD medium (lacks ergosterol).All 20 of the HIS⁺ colonies derived from SWY5-ΔH were able to grow onYPD which indicated that they still contained a functional ERG9 gene,and suggested that the erg9Δ::HIS3 gene was not integrated at the ERG9locus. On the other hand, none of the HIS⁺ colonies derived fromSWE23-ΔHE9 were able to grow on YPD indicating that the erg9Δ::HIS3 DNAhad replaced the wild type ERG9 gene in this strain background at arelatively high frequency. To verify this, nine HIS⁺ colonies of eachstrain were analyzed by PCR to determine the types of ERG9 allelespresent in their genomes. Genomic DNA was prepared as described inSherman et al. (1989). The oligonucleotides used for the analysis aregiven below.

5′ oligo = 250 UpBam (SEQ ID NO: 3) gcgcatCCACGGGCTATATAAA 3′ oligo =1764 LoBam (SEQ ID NO: 4) gcggatCCTATTATGTAAGTACTTAG

PCR conditions were as follows:

94° C., 3 min.

94° C., 30 sec.; 50° C., 30 sec.; 72° C., 3 min.; 30 cycles

72° C., 7 min.

Oligonucleotides 250 UpBam contains sequences corresponding to position#11555 to #11570 of the GenBank Accession #J05091 sequence. Oligo1764LoBam contains the reverse complement of sequences from position#13049 to #13068 of the same GenBank sequence file. In all 9 of the HIS⁺transformants of SWE23-ΔHE9, the PCR product was 2.1 kbp indicating thatthe ERG9 gene had been replaced by the erg9Δ::HIS3 gene. In 8 of the 9HIS⁺ transformants of SWY5-ΔH1, the PCR products were 2.1 kbp and 1.5kbp, indicating that the wild type ERG9 gene remained intact and thatthe erg9Δ::HIS3 DNA had integrated elsewhere in the genome. The ninthHIS⁺ transformant showed only the 1.4 kbp PCR product, also indicatingthat the ERG9 gene was intact in this strain. In this case, the HIS3gene is believed to have integrated into the genome without carrying allof the erg9 sequences with it.

These results show that during the course of isolating strain MBNA1-13,an additional mutation(s), referred to herein as sue, arose in thestrain. Based on the results described in Example 3 below, the suemutation appear to be different from the previously reported upc2mutation and confers more efficient uptake of sterols.

G. Derivation of New Strains from MBNA1-13

In order to use the erg9 mutant strain MBNA1-13 for molecular geneticmanipulations, the strain was further developed so that it carriedauxotrophic markers suitable for this purpose. MBNA1-13 was mutagenizedusing ethyl methane sulfonate using standard procedures and mutantsresistant to 5-fluoroorotic acid (5-FOA) were obtained by plating themutagenized cells on medium containing 5-FOA. The use of 5-FOA selectionwas described in Sikorski and Boeke, 1991 (Sikorski, R. S. and Boeke, J.D., 1991, In Vitro Mutagenesis and Plasmid Shuffling: From Cloned Geneto Mutant Yeast, Methods in Enzymology 194: 302-318). This selectionmethod allowed for the isolation of strains containing mutations in theURA3 gene, coding for orotidine-5′-phosphate decarboxylase. Severalresistant strains were isolated that exhibited 5-FOA resistance (i.e., aura⁻ phenotype). To determine which of these strains contained amutation specifically in the URA3 gene, the ura⁻ strains weretransformed with pRS316 (Sikorski and Hieter, 1989), which contains theintact URA3 gene, using the LiOAc transformation procedure (Gietz etal., 1995). Several strains were identified whose lack of growth onmedium lacking uracil was restored by pRS316, indicating that thosestrains carried the ura3 mutation. One of these strains, EMS9-23 (ura3,erg9) was chosen for further modification.

Strain EMS9-23 was genetically altered to contain deletions in leu2,trp1, or his3 genes using gene transplacement plasmids described inSikorski and Hieter 91989), and the pop-in/pop-out gene replacementprocedure described in Rothstein (1991) (Rothstein, R., 1991, Targeting,Disruption, Replacement, and Allele Rescue: Integrative DNATransformation in Yeast, Methods in Enzymology 194: 281-301). The leu2,trp1 and his3 mutants derived from MBNA 1-13 were named SWE23-ΔL1,SWE23-ΔT1 and SWE23-ΔH1, respectively. Strain SWE23-ΔH1 was furthermanipulated to exchange the original erg9 frameshift mutation (seeSection E) with the erg9Δ::HIS3 allele. This was done by transformingSWE23-ΔH1 (using the LiOAc procedure) with approximately 5 μg of alinear DNA fragment containing the erg9Δ::HIS3 cassette (theconstruction of the erg9Δ::HIS3 cassette is described in detail inExample 3). The erg9Δ::HIS3 cassette was obtained by digesting pKML19-40with BamHI and XmaI, and purifying the 2.1 kbp DNA fragment containingthe erg9Δ::HIS3 fragment. Transformants of SWE23-ΔH1 in which the erg9frameshift allele had been replaced by the erg9Δ::HIS3 allele wereselected by plating the transformants on SCE medium lacking histidine.One of the resulting strains is referred to as SWE23-ΔE91.

Strain SWE23-DH1 was further modified so as to contain mutations in theleu2 and trp1 genes. These mutations were introduced using the genetransplacement plasmids (Sikorski and Hieter, 1981) and thepop-in/pop-out gene replacement procedure (Rothstein 1991) describedabove. One of the resulting strains is referred to as SW23-DH1 #42, andcontains erg9, ura3, his3, leu2, trp1, and sue mutations. StrainSW23-DH1 #42 was further modified to exchange the original erg9frameshift mutation with the erg9D::HIS3 allele. This was done bytransforming SW23-DH1 #42 (using the LiOAc procedure) with approximately5 mg of a linear DNA fragment containing the erg9D::HIS3 cassette asdescribed above. HIS+ transformants in which the erg9 frameshiftmutation had been replaced by the erg9D::HIS3 allele were selected onSCE-his medium. One of the resulting strains is referred to as SW23B,and carries mutations in the following genes: ura3, leu2, trp1, his3,erg9:: HISS, sue.

A summary of the strains derived from MBNA1-13 and their respectivefarnesol production in shake flask cultures is given in Table 6. Thestrains were grown overnight in YPDE medium (30° C. with shaking at 180rpm). These cultures were used to inoculate 25 ml of YPDE medium in a250 ml flask such that the initial OD₆₀₀ was 0.05. The cells were grownfor 72 hours at 30° C. (with shaking at 180 rpm). Samples of the wholebroth (i.e., cells plus culture medium) were analyzed for dry cellweight and farnesol as described in Section C above.

TABLE 6 Growth and Farnesol Production in Strains Derived from MBNA1-13Farnesol Strain Genotype (% of dry cell weight) MBNA1-13 erg9, sue 7.9EMS9-23 erg9, ura3, sue 8.0 SWE23-ΔH1 erg9, ura3, his3-Δ200, sue 7.9SWE23-ΔL1 erg9, ura3, leu2-Δ1, sue 9.3 SWE23-ΔT1 erg9, ura3, trp1-Δ63,sue 10.8 SWE23-ΔE91 erg9Δ::HIS3, ura3, his3, sue 8.0

Strains MBNA1-13, EMS9-23 and SWE23-ΔE91 were further evaluated forgrowth and farnesol production in 1-L fermentors using the methoddescribed in Section H below. EMS9-23 and SWE23-ΔE91 were eachtransformed with plasmid YEp352, which contains the cloned URA3 gene.This obviated the need to add uracil to the fermentation medium forthese two strains. FIG. 2 and Table 7 summarize the results of thesefermentations. MBNA1-13 and EMS9-23/YEp352 performed similarly. Althoughthe growth of SWE23-ΔE91/YEp352 lagged behind the other two strains, itdid reach a cell density and farnesol production level comparable to theother strains.

TABLE 7 Comparison of MBNA1-13 and Strains Derived from it in 1-LFermentors Farnesol Time Dry Cell Weight (% of dry Strain/Plasmid (hr)(g/L) g/L cell weight) MBNA1-13 191 40.8 2.25 5.5 EMS9-23/YEp352 19142.6 2.46 5.8 SWE23-ΔE91/YEp352 239 37.2 2.23 6.0

Strains EMS9-23 and SWE23-ΔH1 were further manipulated by repairingtheir erg9 mutations back to wild type function. This was done bytransforming the two strains (using the LiOAc transformation procedure)with approximately 5 μg of a DNA fragment containing the intact ERG9gene. The ERG9 gene DNA fragment was obtained by digesting pTWM103 (seeSection E above) with Sad and BamHI, and purifying the 2.5 kb DNAfragment containing the ERG9 gene. Transformants of EMS9-23 andSWE23-ΔH1 in which the erg9 frameshift mutation had been replaced by thewild type ERG9 gene were obtained from each strain by selecting forcells that could grow on YPD medium lacking ergosterol. Oneerg9-repaired strain derived from EMS9-23, designated SWE23-E9, and oneerg9-repaired strain derived from SWE23-ΔH1, designated SWE23-ΔHE9, werechosen for further study. In separate experiments, auxotrophic mutationswere introduced into the parental strain ATCC 28383. A spontaneous ura3mutant of ATCC 28383 was obtained after plating cells on mediumcontaining 5-FOA as described above. Mutants resistant to 5-FOA weretransformed with pRS316 (described above) to identify those that hadspontaneously acquired a mutation specifically in their URA3 gene. Onestrain, SWY5, exhibited a stable ura⁻ phenotype (low reversionfrequency), and was chosen for further study. SWY5 was furthermanipulated to carry the his3 mutation by using the gene transplacementplasmid YRp14/his3-Δ200 described by Sikorski and Hieter (Sikorski andHieter, 1989) and the pop-in/pop-out gene replacement proceduredescribed by Rothstein (Rothstein, 1991). One his3 mutant derived fromSWY5, designated SWY5-ΔH, was chosen for further study.

Table 8 lists the repaired and auxotrophic strains described and theirgenotypes.

TABLE 8 Repaired and Auxotrophic Strains related to ATCC 28383 andMBNA1-13 Strain Genotype Parent SWE23-E9 ura3, sue EMS9-23 SWE23-ΔHE9ura3, his3, sue SWE23-ΔH1 SWY5 ura3 ATCC 28383 SWY5-ΔH ura3, his3 SWY5

To test whether the erg9 mutant MBNA1-13 had acquired any additionalnutritional requirements during the course of its isolation, a growthexperiment was conducted in shake flasks to compare the parental strainATCC 28383, the erg9 mutant strain EMS9-23 and the erg9-repaired strainSWE23-E9. The cultures were grown in YPD medium at 30° C. with shakingat 180 rpm. The EMS9-23 culture was also supplemented with 5 mg/Lergosterol. Growth was monitored by OD₆₀₀.

The results of this experiment, presented in FIG. 3, show that thegrowth of the erg9 mutant was reduced, but that the growth of thewild-type parent strain 28383 and the erg9-repaired strain SWE23-E9 grewsimilarly. The slightly lower final OD₆₀₀ in the SWE23-E9 culture wasprobably due to the fact that this strain is a uracil auxotroph, and theconcentration of uracil in the YPD medium was not optimal. These resultsindicate that the major cause of the growth defect in the erg9 mutantsis the erg9 mutation itself. Other mutations in the erg9 mutants, suchas the sue mutation(s), do not have a significant effect on growth.

H. One-Liter Fermentation

Strains MBNA1-13, EMS9-23/YEp352 and SWE23-E91/YEp352 were grown inone-liter fermentors under fed-batch conditions as described below.Under these conditions, farnesol production by these strains ranged from5.5-6.0% of dry cell weight (data shown in Section G above) in 191-239hours.

Fermentation Conditions and Materials

The one-liter fermentation was performed by first autoclaving for 45minutes at 121, in the fermenter, the following solution:

(NH₄)₂SO₄ 10.0 g/L KH₂PO₄ 10.0 g/L CaCl₂—2H₂O 0.5 g/L NaCl 0.5 g/LMgSO₄—7H₂O 3.0 g/L yeast extract (Difco) 2.0 g/L salt solution 20.0 mLdefoamer (Dow 1520) 4.0 drops deionized water 500 mL.

After autoclaving, the following sterile solutions were added:

glucose solution (50%) w/v) 5.0 mL vitamin solution 15.0 mL  ergosterolsolution  0.2 mL.

The glucose and vitamin solutions were sterilized by passage through a0.45 micron filter. The ergosterol solution is considered sterile (seebelow).

Glucose Feed Solution (Made Up in Deionized Water)

glucose solution (60% w/v) 360 mL  yeast extract solution (12.5% w/v) 80mL ergosterol solution 5.8 mL 

Glucose, yeast extract sterilized by autoclaving; ergosterol consideredsterile.

Inoculum

-   -   1 mL cell suspension frozen in 10% glycerol inoculated into 250        mL YPDE medium in 1000 mL baffled flask, incubated 48 hours at        29° C. and 150 rpm; used 30 mL inoculum per fermenter.

Fermentation Conditions

-   -   28° C.    -   pH 4.5    -   Agitation 300-1000 rpm, aeration 50-200 mL/min as required to        maintain dissolved oxygen at 10-60% of air saturation. Glucose        feed solution added after initial glucose was depleted. Addition        rate to achieve a growth rate of 0.04 hr⁻¹ considering that cell        yield on glucose is 0.25. Maximum feed rate=3.5 ml/hr.

Salt Solution (Given Per Liter in Deionized Water)

FeSO₄—7H₂O 0.28 g ZnSO₄—7H₂O 0.29 g CuSO₄—5H₂O 0.08 g Na₂MoO₄—2H₂O 0.24g CoCl₂—6H₂O 0.24 g MnSO₄—H₂O 0.17 g HCl 0.10 mL

Vitamin Solution (Given Per Liter in Deionized Water)

biotin  10 mg Ca-pantothenate 120 mg inositol 600 mg pyridoxine-HCl 120mg thiamine-HCl 120 mg

Ergosterol Solution

ethanol 20 mL IGEPAL 20 mL ergosterol 0.4 g

Heat at 50° C. with mixing until dissolved, store at −20° C.

IGEPAL is 1,3,3 tetramethyl butyl phenoxy(ethoxy)₈ ethanol (SigmaChemical Company, St. Louis, Mo., Product IGEPAL CA-630, Cat. #13021).

Example 2

This example describes the production of erg9 mutants by chemicalmutagenesis of S. cerevisiae strain 64031 using ethylmethane sulfonate(EMS). Strain 64031 (obtained from ATCC) is an erg9-1 mutant thatproduces low levels of farnesol (see Example 1), and additionalmutagenesis produced mutants exhibiting improved yields of farnesol.

A kill curve was performed using EMS, and a mutagenesis of 64031 wasperformed as described in Example 1. Selection was also performed asdescribed in Example 1.

A total of 163 strains were found to be nystatin-resistant, ts, sterolauxotrophs. They were cultured in tubes and shake flasks, and farnesolproduction was determined as described in Example 1. The amounts offarnesol produced in the shake flask cultures are presented in Table 9.

TABLE 9 Farnesol (mg/ml) Farnesol (% dry weight) Strain Cell Medium Cellextracts Medium extracts MBEMS8-1 0.038 0.019 1.7 0.8 MBEMS8-2 0.0480.008 3.6 0.6 MBEMS8-5 0.013 0.022 0.5 1.0 MBEMS8-6 0.045 0.016 5.6 2.0MBEMS8-11 0.024 0.003 2.0 0.2 MBEMS8-12 0.043 0.006 6.9 1.0 MBEMS8-160.026 0.001 3.5 0.2 MBEMS8-18 0.040 0.009 1.4 0.3 MBEMS8-19 0.023 0.0032.8 0.4 MBEMS8-21 0.021 0.009 1.5 0.7 MBEMS8-23 0.025 0.008 2.9 0.9MBEMS8-25 0.018 0.007 1.3 0.5 MBEMS8-26 0.028 0.011 3.7 1.5 MBEMS8-270.035 0.010 5.5 1.5 MBEMS8-31 0.020 0.011 1.1 0.6 MBEMS8-32 0.016 0.0090.5 0.3 MBEMS8-33 0.000 0.000 0 0 MBEMS8-34 0.000 0.000 0 0 MBEMS8-350.000 0.000 0 0 ATCC64031 0.018 0.005 0.9 0.3 ATCC28383 0.000 0.000 0 0

Example 3

In Example 1.G. above, the creation of an erg9Δ::HIS3 mutant of MBNA1-13was described. In this example, the construction of other haploid S.cerevisiae strains (not derived from MBNA1-13) containing an erg9deletion::HIS3 insertion allele is described.

The ERG9 gene was PCR amplified using genomic DNA isolated from strainS288C (according to the method of Sherman et al.) as template and thefollowing two primers:

(SEQ ID NO: 5) 5′ oligo = gag cat CCA CGG GCT ATA TAAA; 250 BamUp(SEQ ID NO: 6) 3′ oligo = tcc ccc cg GGC GCA GAC TTC ACG CTC; 1712 XmaLo

The PCR amplified ERG9 DNA was digested with BamHI and XmaI, thenligated to BamHI, XmaI digested pRS316 (Yeast/E. coli shuttle vector,Sikorski and Hieter, 1989).

The PCR conditions were:

ALIGNL ~94° C.~for~1~min. - ~~~~1~cycle # ALIGNL STACK {~94°C.~for~30~sec.# 58° C.~for~1~min # 72° C.~for~1~min.} ~~~ scalesym 350\}~30~cycles# ALIGNL ~72° C.~for~2~min.-~~~~ 1~cycle

The ERG9 gene cloned in this manner extends from position #11555 toposition #13047 of GenBank Accession #U00030, and the resulting clonewas named pJMB98-12. The construction of the deletion in ERG9 and theinsertion of HIS3 DNA was done as follows:

The HIS3 gene was obtained by digesting the HIS3 containing plasmidpRS403 (Stratagene, LaJolla, Calif.) with Eco47III and SspI, andpurifying the 1226 bp fragment containing the HIS3 gene.

The plasmid pJMB98-12 was digested with Van91I, then treated with T4 DNApolymerase to make it blunt-ended. The plasmid was further digested withHpaI so that an internal 589 bp fragment within ERG9 was deleted. Intothis site was ligated the 1226 bp Eco47III, SspI fragment containing theHIS3 gene. The resulting plasmid, pKML19-41, has the HIS3 gene clonedwithin the deleted ERG9 gene, with the HIS3 coding sequence in the sameorientation as the ERG9 coding sequence.

To transform yeast with this erg9Δ::HIS3 DNA, the plasmid pKML19-41 wasdigested with SmaI and XbaI, and the 2141 bp fragment containing theerg9Δ::HIS3 gene was isolated. DNA was purified from agarose gel slicesby using GeneClean.

Approximately 5 μg of purified erg9Δ::HIS3 DNA was used to transformdiploid strain CJ3A X CJ3B (a/a, ura3/ura3, his3/his3, leu2/leu2,trp1/trp1, upc2/upc2, obtained from Dr. L. Parks, N.C. State University,Raleigh, N.C.) using the lithium acetate transformation proceduredescribed in Gietz, et al. (1995). CJ3A X CJ3B is homozygous for theupc2 mutation. This mutation allows sterol uptake under aerobicconditions (Lewis, et al., 1988). It was believed that the upc2 mutationwould allow the easy production of a haploid strain carrying a mutationin the erg9 gene. The histidine auxotrophy was necessary to select forstrains carrying the plasmids that contain the functional HIS3 gene.

The transformed cells were plated onto SCE medium (described in Example1.F.) lacking histidine (SC-His) to select for HIS transformants.

Hundreds of transformants were obtained. These HIS⁺ cells were thenpatched onto SC-His and allowed to grow for two more days. Thetransformants were then patched onto sporulation medium (Sherman, et.al, 1986). Sporulation medium contains (per liter of distilled water):1% potassium acetate, 0.1% Bacto yeast extract, 0.05% dextrose and 2%Bacto agar. The patches were then allowed to grow and sporulate for 3-5days. A portion of the cells was removed and placed in a solution oflyticase to digest the ascus cell wall. The cells were then spread in athin line onto a YPD+2 mg/L ergosterol (YPDE) plate. The sporulateddiploid cells form tetrads containing four spores, and the individualspores were separated using a micromanipulator. These individual haploidspores will germinate with each containing a single copy of thechromosomes.

A 2:2 segregation was expected for erg9Δ::HIS3 knockouts in this strain.Two spores should be viable on YPD since they would contain the copy ofthe chromosome which was not disrupted (meaning they would not needergosterol since they have a functioning ERG9 gene and would be able togrow on YPD because histidine is present in the medium). The other twospores should grow on SC-His+2 mg/L ergosterol (SCE-His) since, if theerg9Δ::HIS3 DNA integrated at the ERG9 site, the cells would requireergosterol, but would grow without histidine.

Tetrads from some of the pKML19-41 transformants were analyzed forphenotypic determination of the erg9 knockout. The results showed thatonly two of the four spores were viable when grown aerobically. Whenthese were replica-plated to YPD these two spores survived, but they didnot survive on SC-His or SCE-His. This indicated a 2:2 segregation andthat the two surviving spores were of the parental type or auxotrophicfor histidine. This indicated that the two that did not surviveaerobically probably contained the erg9:HIS3 knockout.

PCR analysis confirmed the results of an erg9A::HIS3 knockout in severalof the strains. Total DNA was isolated using a rapid DNA isolationmethod from several of the diploid transformants that showed the 2:2segregation. The total DNA was then used as template for PCRamplification with primers complementing sequence 5′ and 3′ to the ERG9gene in the chromosome.

The oligonucleotides used for this analysis were as follows:5′-oligo=250 Bam Up (described above)

3′-oligo = 3ERG9-1 = (SEQ ID NO: 7)gat ccg cg GCT CAA GCT AGC GGT ATT ATG CC3ERG9-1 contains sequences corresponding to the reverse complement ofsequences from position #14812 to position #14790 of the GenBankAccession #U00030 sequence.

Oligonucleotide 250 Bam Up lies within the sequence of the ERG9 cloneused to construct the erg9::HIS3 allele, whereas oligonucleotide 3ERG9-1lies downstream of the boundary of the cloned ERG9 DNA. ERG9 sequencesamplified from genomic DNA by these two primers must represent ERG9genes located at the actual chromosomal ERG9 locus, and will not amplifyerg9::HIS3 sequences that integrated at other chromosomal locations.

The results showed two bands at 3.271 and 3.908 kb, indicating thepresence in the diploid strains of one functional copy of the ERG9 geneand one interrupted copy that is kb 637 bp larger due to the HIS3 geneinsertion.

Southern blot analysis also was performed to verify the erg9A::HIS3interruption in the diploids. Genomic DNA from a parental diploid straincarrying two normal copies of ERG9 gene was compared to three differenttransformants believed to carry one normal ERG9 gene and one disruptedERG9 gene. The DNA was digested with either Acc I or Xba I, separated bygel electrophoresis, transferred to an Immobilon S membrane, and probedwith specific probes to examine the ERG9 genes present. The probe inthis case was a 1.4 kb fragment containing a portion of the ERG9 gene.

The probe used for Southern blot analysis of genomic DNA was preparedfrom the PCR amplified ERG9 gene using oligonucleotides 250 Bam Up and1712 Xma Lo (described above). Plasmid pJMB98-12 was used as template.

PCR conditions were:

ALIGNL ~94° C.~for~1~min. - ~~~~1~cycle # ALIGNL STACK {~94°C.~for~30~sec.# 58° C.~for~1~min~~ # 72° C.~for~1~min.~} ~~~scalesym350\} ~30~cycles# ALIGNL ~72° C.~~for~2~min. -~~~~1~cycle

The amplified ERG9 DNA was purified and biotinylated, as describedabove. Approximately 1 μg of biotinylated probe was used to hybridize tothe blot.

Hybridization of the probe to the specific DNA bands under stringentconditions identified complementary sequence being present. For theparental diploid, CJ3AxCJ3B, a single band was seen at 2.1 kb for theAcc I digest and a single band at 2.8 kb was seen with the Xba I digest.The putative erg9Δ::HIS3 knockouts each contained two bands: bands at2.1 kb and 1.3 kb for the Acc I digest; and bands at 2.8 kb and 3.4 kbfor the Xba I digest. These results indicated the presence of one copyof the wild-type ERG9 gene along with an erg9Δ::HIS3 disruption in thetransformed diploids.

Attempts were then made to grow the haploid cells anaerobically on YPDEagar. Strains that are blocked in the ergosterol pathway can take upsterols under anaerobic conditions. Two of the spores grew rapidly underanaerobic conditions (ERG9, his-) and two grew very slowly(erg9Δ::HIS3). The spores of three of the strains erg9Δ::HIS3 (KML505,KML510, and KML521) that grew anaerobically on plates were grown inliquid medium under anaerobic conditions and showed farnesol production.Upon further incubation of the original YPDE plates aerobically, itappeared that a few of the haploid strains that tested positive forfarnesol production under anaerobic growth were growing. Three suchviable spores (derived from KML505) arose after prolonged aerobicincubation. These aerobically-growing strains, designated as BKY1-2D,BKY1-7C, and BKY1-8D, all produced farnesol under aerobic conditions.

The parent strain KML510 (a/a, ura3/ura3, his3/his3, trp1/trp1,leu2/leu2, upc2/upc2, ERG9/erg9Δ::HIS3) containing either pJMB98-12 orpRS316 were sporulated and tetrads were dissected onto YPDE plates. Theplates were incubated under aerobic conditions, and the followingpatterns of spore growth were observed.

Tetrad analysis of KML510/pJMB98-12 were done with 20 tetrads. 16tetrads gave rise to two viable, fast-growing spores and two nonviablespores. All viable spores were his⁻, erg⁺. Three tetrads gave rise totwo viable, fast-growing spores, one viable, slow-growing spore, and onenon-viable spore. The viable, fast-growing were all his⁻, erg⁺. Theviable, slow-growing spores were all his⁺, erg⁻. One tetrad did not giverise to any viable spores. All of the his⁺, erg⁻ spores were also ura⁻,indicating that pJMB98-12 did not segregate into these spores duringmeiosis. One of the his⁺, erg⁻ spores was used for further studies, andwas named BTY19-9C.

Tetrad analysis of KML510/pRS316 was done with 20 tetrads. 18 tetradsgave rise to two viable, fast-growing spores and two non-viable spores.Two tetrads gave rise to two viable, fast-growing spores, one viable,slow-growing spore, and one non-viable spore. All fast-growing sporeswere his⁻, erg⁺. All slow-growing spores were his⁺, erg⁻. All his⁺, erg⁻spores were also ura⁻, indicated that pRS316 did not segregate intothese spores during meiosis. One of the his⁺, erg⁻ spores was studiedfurther, and was named BTY20-2A.

To confirm that BTY19-9C and BTY20-2A did not carry the wild-type ERG9gene, a PCR experiment was conducted using genomic DNA isolated from thetwo strains, and the oligonucleotides VE104-5 and VE105-3.

5′oligo = VE104-5 = gac tct AGA AGC GGA AAA CGT ATA CAC 3′oligo =VE105-3 = described above

VE104-5 contains sequences corresponding to position #11631 to 116541 ofGenBank Accession #U00030 sequence.

The predicted PCR product size for a functional ERG9 gene was 2.23 kb,and for the erg9Δ::HIS3 disruption, 2.87 kb. Strains BTY19-9C andBTY20-2A had a PCR product of approximately 2.87 kb, which indicated thepresence of the erg9Δ::HIS3 disruption.

Five strains (BKY1-2D, BKY1-7C, BKY1-8D, BTY19-9C and BTY20-2A) wereevaluated for farnesol production in shake flasks. Strains BKY1-7C andBTY20-2A gave the best production of farnesol of the five strains, butBKY1-7C grew very slowly.

Transformants KML505, KML510, and KML521 were transferred to sporulationmedium. Tetrads were dissected onto YPDE and allowed to grow forextended periods of time under aerobic conditions to look for growth ofadditional spores beyond the anticipated 2:2 segregation(viable:nonviable). Two additional spores were viable after furtherincubation under aerobic conditions. These two strains, BJY7-5D andBJY8-1A, produced farnesol in a tube screen.

Strains BJY7-5D and BJY8-1A were evaluated in shake flasks. BJY8-1A gavethe best farnesol production (0.29 mg/ml, 4.7% based on cell dry weight)after 72 hours incubation. BJY7-5D gave 0.18 mg farnesol/ml, (3.5% basedon cell dry weight).

Example 4

This example shows the overexpression of HMG CoA reductase in strainswith and without a functional ERG9 gene.

HMG CoA reductase has been proposed to be the key enzyme regulating theflow of carbon through the isoprenoid pathway. In S. cerevisiae, twogenes, HMG1 and HMG2, code for the two isozymes of HMG CoA reductase,designated HMG1p and HMG2p. Regulation of HMG CoA reductase is achievedthrough a combination of transcriptional and translational regulation aswell as protein degradation. The segments of the HMG1 and HMG2 genesthat encode the catalytic domains of the HMG1p and HMG2p isozymes havebeen cloned under transcriptional control of the strong promoter fromthe GPD (glyceraldehyde-3-phosphate dehydrogenase) gene. The plasmidscontaining these constructions (pRH127-3 and pRH124-31, containing thecatalytic domains of HMG1p and HMG2p, respectively) were obtained fromR. Hampton, U.C. San Diego. Strains of S. cerevisiae overexpressing thecatalytic domain of HMG1p were reported to have an elevated flow ofcarbon through the isoprenoid pathway. This increased carbon flow wasmanifested as squalene and ergosterol overproduction (Donald, K. A. G.,Hampton, R. Y., and Fritz, I. B., 1997, Effects of Overproduction of theCatalytic Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase onSqualene Synthesis in Saccharomyces cerevisiae. Applied andEnvironmental Microbiology 63:3341-3344).

A. HMG CoA Reductase Overexpression in Strains with a Functional ERG9Gene

In order to confirm that overexpressing HMG CoA reductase would resultin increased carbon flow through the isoprenoid pathway in the strainsderived from ATCC 28383, similar to other strains of S. cerevisiae(Donald, et. al., 1997), the strain SWY5, a ura3 mutant of the parentalstrain ATCC 28383, and SWE23-E9, the erg9 repaired version of EMS9-23(strains described in Example 1.G. above), were transformed withplasmids pRH127-3 and pRH124-31. Transformations were performed usingthe LiOAc procedure (Gietz, et. al., 1995). Control strains carrying theempty vector YEp352 were also constructed. Representative transformantswere tested for squalene and ergosterol production in a shake flaskexperiment. Cells were grown in 50 ml SCE-ura medium at 30° C., withshaking at 180 rpm for 48 hours. At 24 hours, 10 ml of the culture waswithdrawn, and the cells harvested for HMGCoA reductase assays asdescribed in Example I.D. At the end of the 48-hour growth period, analiquot of the cultures was used to inoculate flasks containing 50 mlYPD medium such that the starting OD₆₀₀ of the cultures was 0.1. Thecultures in YPD medium were grown for 72 hours at 30° C., with shakingat 180 rpm, and then analyzed for dry cell weight, squalene andergosterol accumulation. The extraction method used for this analysiswas as follows. Ten ml of the culture was pelleted by centrifugation at1,500×g for 10 minutes. The cell pellet was resuspended in 1 ml ofdeionized water, and the cells were repelleted. Cell pellets wereresuspended in 1 ml of deionized water and disrupted by agitation withzirconium silicate beads (0.5 mm diameter). The broken cell suspensionwas saponified with 2.5 ml of a 0.2% solution of pyrogallol (inmethanol) and 1.25 ml of 60% potassium hydroxide solution at 75 degreesC. for 1.5 hours. The samples were then extracted with 5 ml of hexane,vortexed for 3 minutes and centrifuged at 1,000×g for 20 minutes toseparate the phases. The hexane layer was then analyzed by GC-MS asdescribed in Example 1.B.

The data from this experiment are shown in Table 10. Compared to thecontrol strains carrying only the Yep352 vector, the strains thatoverexpress the catalytic domains of either HMG1p or HMG2p containedhigh levels of HMGCoA reductase activity and elevated levels ofsqualene. However, neither of the strains overexpressing HMG1p or HMG2pcontained significantly increased levels of ergosterol. Nevertheless,these data show that increasing the activity of HMG CoA reductase in theMBNA1-13 strain lineage increases the carbon flow through the isoprenoidpathway.

TABLE 10 Effect of Amplified HMGCoA Reductase Activity on Squalene andErgosterol Production in Strains Having a Normal Isoprenoid Pathway HMGCoA Reductase Dry Cell Squalene Ergosterol Activity Strain/ Weight % dry% dry (nmol/min/ plasmid (g/L) μg/ml wt. μg/ml wt. mg) SWY5/ 7.68 0.180.003 2.9 0.038 2.4 YEp352 SWY5/ 6.82 3.50 0.051 5.2 0.076 52 pRH127-3SWY5/ 7.49 4.15 0.055 2.9 0.039 27 pRH124-31 SWE23-E9/ 6.96 0.33 0.0055.5 0.079 2.7 YEp352 SWE23-E9/ 6.89 6.78 0.099 8.3 0.121 55 pRH127-3SWE23-E9/ 6.90 4.90 0.071 6.1 0.088 29 pRH124-31B. HMG CoA Reductase Overexpression in an erg9 Mutant

Having shown that amplification of HMG1p or HMG2p increased carbon flowto squalene, whether overexpression of HMG/or HMG2 would increasefarnesol production in a strain carrying the erg9 mutation was tested.Strain SWE23-ΔE91 (described in Example 1.G.) was transformed withplasmids pRH127-3 or pRH124-31. Transformation of SWE23-ΔE91 wasaccomplished using the LiOAc transformation procedure (Gietz et al,1996). Approximately 2.5 μg of pRH127-3 or pRH124-31 DNA were used ineach transformation. Transformants were selected on SCE-ura plates, andseveral were chosen and restreaked for purification on SCE-ura plates.To test the effect of amplified HMG CoA reductase on farnesolproduction, representative transformants were grown for 48 hours inliquid SCE-ura medium. A control strain, SWE23-ΔE91/YEp352, was alsoincluded in the experiment. At the end of the 48 hours growth period,aliquots of the cultures were used to inoculate flasks containing 50 mlof YPDE medium such that the initial OD₆₀₀ was 0.5. These cultures weregrown for 72 hours at 30° C., with shaking at 180 rpm. At the end of theincubation period, samples were analyzed for dry cell weight andfarnesol concentration. To confirm that the HMG CoA reductase genes werebeing overexpressed in the transformants, 20 ml of the cultures grown inSCE-ura were harvested by centrifugation at 1500×g for 10 minutes, andHMG CoA reductase activity measured using the permeabilized cell methoddescribed in Example I.D. The data from this experiment are shown inTable 11.

TABLE 11 Effect of Elevated HMG CoA Reductase Activity on FarnesolProduction in an erg9:.-HIS3 Mutant Grown in Shake Flask Cultures. DryCell HMG CoA Reductase Weight Activity Strain/plasmid (g/L) Farnesol(nmol/min/mg) SWE23-ΔE91/YEp352 3.31 222 6.7 7.5 SWE23-ΔE91/ 3.87 1042.7 47.0 pRHI27-3 SWE23-ΔE91/ 3.48 104 3.0 36.0 pRH124-31

In both SWE23-ΔE91/pRH127-3 and SWE23-ΔE91/pRH124-31, the levels of HMGCoA reductase activity were elevated. However, the farnesol productionin these strains was lower than the control strain SWE23-ΔE91/YEp352.This unexpected result was observed several times in independentexperiments.

Despite the failure of amplified HMG CoA reductase activity to increasefarnesol production in an erg9Δ::HIS3 strain grown in shake flaskcultures, the strains listed in Table 12 were tested for farnesolproduction in 1-L fermentors using the protocol described in Example1.H. Under these conditions, the expected result was observed; thestrains overexpressing HMG CoA reductase produced significantly morefarnesol than the control strain. The data are summarized in FIG. 4 andTable 12. The elevation of HMG CoA reductase activity in strainsSWE23-ΔE91/pRH127-3 and SWE23-ΔE91/pRH124-31 was confirmed by directenzyme assay using the permeabilized cell method. This is illustrated bythe reductase activities at specific time points during the fermentation(shown in Table 12).

TABLE 12 Effect of Elevated HMG CoA Reductase Activity on FarnesolProduction in an erg9Δ::HIS3 Mutant Grown in 1-L Fermentors. FarnesolHMG CoA Time Dry Cell % dry cell Reductase Activity Strain/plasmid (hr.)Weight (g/L) g/L weight (nmol/min/mg) SWE23-ΔE91/YEp352 239 37.2 2.236.0 4.1 SWE23-ΔE91/pRHI27-3 310 35.0 3.06 8.7 20 SWE23-ΔE91/pRH124-31261 31.7 3.05 9.6 14

Example 5

This example describes the overexpression of various GGPP synthases inerg9 mutants.

In order to convert FPP produced in erg9 mutant strains to GGPP (theprecursor of desired product, GG), the genes coding for various GGPPsyntheses in erg9 mutants were overexpressed. The genes cloned for thispurpose were the BTS1 gene from Saccharomyces cerevisiae, the crtE genefrom Erwinia uredovora, the al-3 gene from Neurospora crassa, and theggs gene from Gibberella fujikuroi. Plasmids carrying these genes wereconstructed as follows.

To clone the BTSI1 gene, genomic DNA was prepared from S. cerevisiaestrain S288C according to the method described in Sherman, et. al.(1986). The gene was amplified by PCR using the following twooligonucleotides.

5′oligo = VE100-5 (SEQ ID NO: 8) gactctAGAGTTTACGAGTCTGGAAAATC 3′oligo =VE101-3 (SEQ ID NO: 9) gtaggATCCATGGAATTGAGCAATAGAG

The PCR conditions were: 94° C., 3 minutes; 94° C., 1 minute; 52° C., 1minute; 74° C., 1.5 minutes; 30 cycles; then 74° C., 7 minutes (1cycle). The PCR product was digested with XbaI and BamHI and ligatedinto XbaI, BamHI digested pPGK315 to generate pTWM101. The plasmidpPGK315 contains the S. cerevisiae PGK promoter and terminator separatedby a multiple cloning site. Cloning the BTS1 gene into pPGK315 asdescribed above placed the BTS1 gene under control of the strong PGKpromoter. Plasmid pPGK315 was constructed by digesting plasmid pRS315with SacII and SmaI, then treating with T4 DNA polymerase to createblunt ends. The plasmid was religated to generate pRS315-DSS. PlasmidpRS315DSS was then digested with XhoI and ligated with a 994 bp SaII,XhoI fragment obtained from plasmid pPGKMCS that contained the PGKpromoter and terminator separated by a multiple cloning site. PlasmidpPGKMCS was constructed by digesting pPGK (Kang et. al., 1990, Mol.Cell. Biol., 10:2582) with EcoRI and BamHI, then ligating with thefollowing two annealed oligonucleotides.

(SEQ ID NO: 10) 5′oligo = AATTCCAAGCTTGCGGCCGCTCTAGAACGCGTG(SEQ ID NO: 11) 3′oligo = GATCCACGCGTTCTAGAGCGGCCGCAAGCTTGPlasmid pTWM110-11 was constructed by ligating a 2397 bp KpnI-SalIfragment containing the PGK promoter/BTS1/PGK terminator (obtained bydigestion of PTWM110 with KpnI and SalI) into KpnI-Sa/1-digested YEp352.

To clone the crtE gene, genomic DNA was isolated from E. uredovorastrain 2OD3 using the PureGene genomic DNA Isolation Kit (GentraSystems, Inc., Minneapolis, Minn.) The crtE gene was amplified using thegenomic DNA and the following two oligonucleotides.

5′oligo = 20D3Up (SEQ ID NO: 12) gaattcGTTTATAAGGACAGCCCGA 3′oligo =20D3Lo (SEQ ID NO: 13) ctgcagTCCTTAACTGACGGCAGCGA

Oligo 2OD3Up contains sequences corresponding to position #206 to #224of the Genbank Accession #D90087 sequence. Oligo 20D3Lo contains thereverse complement of sequences from position #1117 to #1136 of the sameGenbank sequence file. The amplified DNA was ligated into the SrfI siteof pCR-Script SK(+) (Stratagene, La Jolla, Calif.) to generate plasmidpSW1-B. This plasmid was then digested with EcoRI and SacI, and the 973bp fragment containing the crtE gene was purified and ligated intoEcoRI-SacI digested pAD314-956 to generate plasmid pSW2B. Plasmid pSW2-Bwas digested with BamHI to generate a 2199 bp fragment containing theADH1 promoter, crtE coding sequence, and ADH1 terminator, and thisfragment was ligated into BamH digested YEp352 to generate pSW4A, whichplaced the crtE gene in the orientation opposite to that of the URA3gene in the plasmid, and pSW4B, which placed the crtE gene in the sameorientation as the URA3 gene.

Plasmid pADH313-956 was generated in the following manner. PlasmidpRS313 (Sikorski and Hieter, 1989) was digested with SacI and XbaI,treated with T4 DNA polymerase to create blunt ends, then relegated togenerate plasmid pDSX313. This plasmid was digested with SmaI and ApaI,treated with T4 DNA polymerase to create blunt ends, and then relegatedto generate pDSXSA313. Plasmid pAAH5 (Ammerer, G., 1983, Meth. Enzymol.101:192-201), which carries the ADH1 promoter and terminator separatedby a HindIII site, was digested with HindIII, then ligated to thefollowing two annealed oligonucleotides to introduce a multiple cloningsite and create pAAH5-MCS.

5′ oligo = MCS1 (SEQ ID NO: 14)AGCTGAATTCGAGCTCGGTACCCGGGCTCTAGAGTCGACCTGCAGGCAT GCA 3′ oligo = MCS2(SEQ ID NO: 15) AGCTTGCATGCCTCCAGGTCGACTCTAGAGCCCGGGTACCGAGCTCGAA TTC

A PCR reaction containing pADH313-MCS as template and the following twooligonucleotides was performed, and a 1232 bp PCR product containing theADH1 promoter, multiple cloning site, and ADH1 terminator was obtained.

5′oligo = ADH1A (SEQ ID NO: 16) gacggatCCGTGGAATATTTCGGATATCC 3′oligo =ADH1B (SEQ ID NO: 17) ctcggatccGGACGGATTACAACAGGTATTGTCC

Oligo ADH1A contains sequences corresponding to position #16 to #37 ofthe Genbank Accession #V01292 sequence. Oligo ADH1B contains the reversecomplement of sequences from position #2092 to #2116 of the same Genbanksequence file. The 1232 bp PCR product was digested with BamHI andligated into BamHI digested pDSXSA313 to generate plasmid pADH313-956.

To clone the N. crassa al-3 gene, genomic DNA was isolated from N.crassa strain ATCC 14692 using the method described by Borges et al. inthe methods section of the Fungal Genetics Stock Center's web site(www.kumc.edu/research/fgsc/fgn37/borges.html). The gene was amplifiedby PCR using the following two oligonucleotides.

5′oligo = VE118-5 (SEQ ID NO: 18) cagaatTCACCATGGCCGTGACTTCCTCCTC3′oligo = VE119-3 (SEQ ID NO: 19) caagatctCATACATTCAATCCTCATGGACAC

Oligo VE118-5 contains sequences corresponding to position #1216 to#1240 of the Genbank Accession #U20940 sequence. Oligo VE119-3 containsthe reverse complement of sequences from position #2576 to #2599 of thesame Genbank sequence file. The amplified DNA was ligated into the SrfIsite of pCR-Script SK(+) (Stratagene, La Jolla, Calif.) to generatepSW7-1 and pSW7-2, two independent PCR clones of the al-3 gene. Thesetwo plasmids were then digested with EcoRI and BglII, and the 1393 bpfragment containing the al-3 gene was purified and ligated into EcoRI,BamHI digested pPGK (Kang et. al., 1990) to generate pSW9-1 and pSW9-2,with the al-3 sequence in each derived from pSW7-1 and pSW7-2,respectively.

To clone the ggs gene, genomic DNA was prepared from Gibberella fujikuoistrain ATCC 12617, using the same procedure described above forNeurospora. The ggs gene was amplified by PCR using the following twooligonucleotides.

5′oligo = VE120-5 (SEQ ID NO: 20) gagaattCTTAACACGCATGATCCCCACGGC3′-oligo = VE121-3 (SEQ ID NO: 21) ctggatcCGTCAAATCCGTGAATCGTAACGAG

Oligo VE120-5 contains sequences corresponding to position #607 to #630of the Genbank Accession #X96943 sequence. Oligo VE121-3 contains thereverse complement of sequences from position #1908 to #1932 of the sameGenbank sequence file. The amplified DNA was ligated into the SrfI siteof pCR-Script SK(+) to generate pSW8-1 and pSW8-2, two independent PCRclones of the ggs gene. These two plasmids were then digested with EcoRIand BamHI, and the 1335 bp fragment containing the ggs gene was purifiedand ligated into EcoRI, BamHI digested pPGK to generate pSW10-1 andpSW10-2, with the ggs sequence in each derived from pSW8-1 and pSW8-2,respectively.

Strain EMS9-23 (ura3, erg9, described in Example 1.G.) was transformedwith the plasmids described above using the LiOAc method, andtransformants were selected on SCE-ura plates. Transformants were pickedand restreaked on SCE-ura plates for purification. Representativetransformants were tested for GG production. The strains were grown at30° C. for 48 hours in liquid SCE-ura medium, then used to inoculateflasks containing YPDE medium, such that the initial OD₆₀₀ was 0.5.These cultures were incubated at 30° C. with shaking for 72 hours andanalyzed for dry cell weight, farnesol and GG levels. A second set offlasks containing 20 ml of SCE-ura medium was also inoculated from thesame starting cultures and was grown for 48 hours. The cells from theseflasks were harvested, washed with 10 ml 50 mM BisTris-Propane buffer,pH 7.0, and repelleted. The cell pellets were then used to preparepermeabilized cell suspensions for GGPP synthase assays. The cells werepermeabilized by resuspending in 1 ml of 50 mM Bis-Tris-Propane buffer,pH 7.0, containing 0.1% Triton X100, and frozen at 80° C. until needed.After thawing, the permeabilized cells were used for GGPP synthaseassays. The GGPP synthase assay mixture contained 0.05M Bis-Tris propanebuffer, pH 7.0 (X μl), 0.1M dithiothreitol (1 μl), 1 mg/ml FPP (5 μl), 1mg/ml IPP (5 μl) and permeabilized cells (Y μl), where X+Y=87 μl. Acontrol reaction was included in which the FPP and IPP were omitted. Theassay mixtures were incubated at 37° C. for 20 minutes. 0.1 ml of 2×glycine buffer (0.2M glycine, 2 mM MgCl₂, 2 mM ZnCl₂, pH 10.4) and 63units of alkaline phosphatase (Sigma Chemical Co., St. Louis, Mo.) wereadded, and the mixtures were incubated for 60 minutes at 37° C. Themixtures were then extracted with 0.2 ml 1:1 hexane:ethyl acetate, andanalyzed for GG by GC/MS. GGPP synthase activity is expressed as nmolGGPP formed/min/mg protein.

The dry cell weights, farnesol production, GG production and GGPPsynthase activities for the strains are summarized in Table 13. Thecontrol strain, EMS9-23/YEp352, which lacks a cloned GGPP synthase, made8.7% farnesol (based on dry cell weight) and essentially no GG. The fourother strains, each of which overexpressed a different cloned GGPPsynthase, produced approximately the same amount of farnesol as thecontrol (8.0-9.74%) and produced levels of GG ranging from 0.62-1.73%.

TABLE 13 GG Production in Shake Flask Cultures of Strains OverexpressingFour Different Cloned GGPP Synthase Genes. GGPP Cloned FOH GC synthaseGGPP Dry cell (% of (% of activity synthase weight dry cell dry cell(nmol/ Strain gene (g/L) weight) weight) min/mg) EMS9-23/YEp352 — 3.388.7 0.01 ND EMS9- BTS1 3.12 8.98 1.73 0.065 23/pTWM110-11 EMS9-23/pSW4BcrtE 2.02 9.74 1.68 0.094 EMS9-23/pSW9-1 al3 3.38 8.0 0.62 0.032EMS9-23/pSW10-2 ggs 3.16 9.3 1.0 0.03 

The effect of amplification of GGPP synthase activity on GG productionwas further evaluated in I-L fermentors. Strain EMS9-23 was transformedwith plasmid pTWMI10-11, carrying the cloned S. cerevisiae BTS1 gene.Strain SWE23-ΔE91 (described in Example 1.G. above) was transformed witheither Yep352 (vector control) or pSW4A-3 (an individual isolate ofplasmid pSW4A, carrying the cloned crtE gene). StrainsSWE23-ΔE91/YEp352, EMS9-23/pTWM110-11 and SWE23-ΔE91/pSW4A-3 were grownin 1-liter fermentors using the method described in Example 1.H., andgrowth, farnesol production and GG production were compared.

The results are shown in FIG. 5 and Table 14. The growth of the threestrains was similar. Strain SWE23-ΔE91/YEp352 produced 2.32 g/L farnesolin 236 hours, and did not produce any detectable GG. EMS9-23/pTWM110-11produced 2.1 g/L farnesol in 239 hours and also produced 0.42 g/L GG inthe same time period. Strain SWE23-ΔE91/pSW4A-3 produced 2.47 g/Lfarnesol in 236 hours and also produced 0.59 g/L GG in the same period.The GGPP synthase activity in the control strain was below detectablelimits. The production of GG by strains EMS9-23/pTWM110-11 andSWE23-ΔE91/pSW4A-3 correlated to increased GGPP synthase activity.

TABLE 14 Growth, Farnesol and GG Production in Strains OverexpressingGGPP Synthases and Grown in 1-L Fermentors GGPP Dry Farnesol GG SynthaseCell % Dry % Dry Activity Strain/ Time Weight Cell Cell (nmol/min/Plasmid (hr.) (g/L) g/L Weight g/L Weight mg) SWE23-ΔE91/ 236 43.9 2.325.3 0 0 not detected YEp352 EMS9-23/ 239 41.9 2.10 5.0 0.42 1.0 0.2pTWM110-11 SWE23-ΔE91/ 236 43.5 2.47 5.7 0.59 1.4 1.1 pSW4A-3

Example 6

This example illustrates the effect of overexpressing the ERG20 genewhich encodes FPP synthase.

To determine the effects of elevating FPP synthase levels in erg9mutants, the ERG20 gene coding for FPP synthase from Saccharomycescerevisiae was cloned for overexpression. The ERG20 gene was amplifiedby PCR using genomic DNA from S. cerevisiae strain S288C (preparedaccording to the method described in Sherman et al., 1986), and thefollowing two oligonucleotides.

(SEQ ID NO: 22) 5′oligo = BamFPPUp ggccggatccATATTACGTAGAAATGGCTT CAG(SEQ ID NO: 23) 3′oligo = XhoFPPLo gccgctcgagGGTCCTTATCTAGTTTG

Oligo BamFPPUp contains sequences corresponding to position #790 to #810of the Genbank Accession #J05091 sequence. Oligo XhoFPPLo contains thereverse complement of sequences from position #1891 to #1907 of the sameGenbank sequence file. The PCR product was digested with BamHI and XhoI,and ligated into a vector containing the S. cerevisiae GPD(glyceraldehyde-3-phosphate dehydrogenase) promoter and PGK terminator.The promoter/terminator vector was obtained by digesting a plasmidcontaining the catalytic domain of the S. cerevisiae HMG2 gene clonedbetween the GPD promoter and PGK terminator, namely pRH124-31 (describedin Example 4), with BamHI and SalI to remove the HMG2 DNA. The 6.3 kbband corresponding to the promoter/terminator vector was purified andligated to the 1129 bp fragment containing ERG20 to generate pJMB1931and pJMB19-32. Plasmids pJMB19-31 and pJMB19-32 were used to transformstrain SWE23-ΔE91 (ura3, his3, erg9Δ::HIS3) using the LiOAc method, andtransformants were selected on SCE-ura plates. Transformants were pickedand restreaked on SCE-ura plates for purification.

To determine the effects of FPP synthase overexpression in erg9 mutantstrains, representative transformants constructed as described abovewere grown in shake flask cultures and compared for growth, farnesolproduction, GG production and FPP synthase and GGPP synthase activities.The strains were grown with shaking at 30° C. for 48 hours in liquidSCE-ura medium, then used to inoculate flasks containing YPDE medium,such that the initial OD₆₀₀ was 0.5. These cultures were incubated at30° C. with shaking for an additional 72 hours and analyzed for dry cellweight, farnesol and GG levels. A second set of flasks containing 20 mlof SCE-ura medium was also inoculated from the same starting culturesand was grown for 48 hours. The cells from these flasks were harvested,washed with 10 ml 50 mM BisTris-Propane buffer, pH 7.0, and repelleted.The cell pellets were then used to prepare permeabilized cellsuspensions for FPP and GGPP synthase assays. The cells werepermeabilized by resuspending in 1 ml of 50 mM Bis-Tris-Propane buffer,pH 7.0, containing 0.1% Triton X100, and frozen at 80° C. until needed.After thawing, the permeabilized cells were used for assays. The GGPPsynthase assay mixture was the same as described in Example 5. The FPPsynthase assay mixture contained 0.05 M Bis-Tris propane buffer, pH 7.0(X μl), 0.1M dithiothreitol (1 μl), 0.5M MgCl₂ (2 μl), 1 mg/ml IPP (6μl), 1 mg/ml geranyldiphosphate (GPP) (6 μl), and permeabilized cells (Yμl), where X+Y=85 μl. A control reaction was included in which the IPPand GPP were omitted. The assay mixtures were incubated at 37° C. for 15minutes. 0.1 ml of 2× glycine buffer (0.2M glycine, 2 mM MgCl₂, 2 mMZnCl₂, pH 10.4) and 63 units of alkaline phosphatase (Sigma ChemicalCo., St. Louis, Mo.) were added, and the mixtures were incubated for 60minutes at 37° C. The mixtures were then extracted with 0.2 ml 1:1hexane:ethyl acetate, and analyzed for Farnesol by GC/MS. FPP synthaseactivity is expressed as nmol FPP formed/min/mg protein.

The data presented in Table 15 show that overexpression of FPP synthasedid not increase farnesol production, but did, unexpectedly, increase GGproduction in these shake flask cultures. The level of GG produced bySWE23-ΔE91/pJMB19-31 or SWE23-ΔE91/pJMB19-32 was even higher thanobserved for the strain (EMS9-23/pTWM110-11) overexpressing the S.cerevisiae BTS1 (GGPP synthase) gene.

TABLE 15 GG production in erg9 mutants overexpressing FPP synthase andGGPP synthase FPP GGPP Cloned FOH (% GG (% synthase synthase gene Drycell of dry of dry activity activity Strain/ (enzyme weight cell cell(nmol/min/ (nmol/min/ plasmid encoded) (g/L) weight) weight) mg) mg)EMS9-23/ — 3.32 9.24 0.17  2.3 Not YEp352 detected EMS9-23/ BTS1 (GGPP3.12 8.98 1.73 Not 0.19 pTWM110-11 synthase) tested SWE23-ΔE-91/ ERG20(FPP 3.11 7.85 2.28 53.0 Not pJMB19-31 synthase) tested SWE23-ΔE-91/ERG20 (FPP 3.28 8.08 2.29 51.0 Not pJMB19-32 synthase) tested

The effect of amplification of FPP synthase activity on GG productionwas further evaluated in 1-L fermentors. Strains SWE23-ΔE91/YEp352(control), EMS9-23/pSW4A3 (crtE, GGPP synthase) and SWE23-ΔE91/pJMB19-32(ERG20, FPP synthase) were grown in 1-liter fermentors using the methoddescribed in Example 1.H., and growth, farnesol production, GGproduction and GGPP synthase activity were compared (FIG. 6 and Table16). The growth of the three strains was similar, althoughSWE23-ΔE91/pJMB19-32 lagged behind before growing at a rate similar tothe other strains. Strain SWE23-ΔE91/YEp352 produced 2.31 g/L farnesolin 258 hours, and did not produce any detectable GG. EMS9-23/pSW4A3produced 2.52 g/L farnesol and 0.57 g/L GG in 258 hours. StrainSWE23-ΔE91/pJMB19-32 produced 1.95 g/L farnesol in 258 hours and alsoproduced 0.59 g/L GG in the same period. The unexpected GG production bySWE23-ΔE91/pJMB19-32 correlated to increased GGPP synthase activity inthis strain (see below).

No GGPP synthase activity was detected in the control strainSWE23-ΔE91/YEp352. Strain EMS9-23/pSW4A3, expressing the E. uredovoracrtE (GGPP synthase) gene showed relatively high GGPP synthase activity(1.7 nmol/min/mg in cells from the 258-hour time point). As expected,strain SWE23-ΔE91/pJMB19-32, which overexpresses the S. cerevisiae FPPsynthase, contained FPP synthase activity that was elevated >10-fold atall time points across the fermentation compared to a control havingonly normal levels of FPP synthase (data not shown). Surprisingly,however, strain SWE23-ΔE91/pJMB19-32 also had elevated GGPP synthaseactivity (Table 16). This activity undoubtedly accounts for theunexpected GG production by this strain.

The elevated GGPP synthase activity detected in strainSWE23-ΔE91/pJMB19-32 could be a result of the overexpression of FPPsynthase inducing the activity of the GGPP synthase (the BTS1 geneproduct). It is also possible, however, that the FPP synthase has somelow level of GGPP synthase activity which is revealed in this strainbecause of the high degree of overproduction of the FPP synthase.

TABLE 16 Effect of Amplified FPP Synthase on Growth, Farnesol and GGProduction of Strains Grown in 1-L Fermentors GGPP Farnesol GG SynthaseDry Cell % of Dry % of Dry Activity Strain/ Time Weight Cell Cell(nmol/min/ plasmid (hr.) (g/L) g/L Weight g/L Weight mg) SWE23-ΔE91/ 25842.2 2.31 5.5 0 0 Not detected Yep352 EMS9-23/ 258 44.3 2.52 5.3 0.571.28 1.7 pSW4A3 SWE23-ΔE91/ 258 41.1 1.95 4.7 0.55 1.34 1.0 pJMB19-32

Example 7

This example evaluates several farnesol extraction procedures.

In the first experiment, a comparison was made between extraction of thewhole broth (cells plus medium) and separate extractions of the cellpellet and medium under various conditions for MBNA1-13 (farnesolproducer) and ATCC 28383 (ergosterol producer) (see Example 1). A 10 mlsample of culture was extracted generally as described in Example 1, butwith the changes indicated in Table 17a below. Method 1 in Table 17a isthe same as the extraction method described in Example 1. It was foundthat, for farnesol extraction, a whole broth extraction was just asefficient as a separate cell and medium extraction for methods 1, 2, 3,5 and 7. The most farnesol was extracted by method 5, which is just a 2ml addition of 0.2% pyrogallol in methanol prior to extracting the wholebroth into hexane. The second highest extraction was method 1.

TABLE 17a Extraction pyrogallol 60% KOH Incubation method 0.2% in MeOHin dH2O 75°, 1.5 h Hexane 1 2 ml 1 ml yes 5 ml 2 2 ml 1 ml no 5 ml 3 2ml 0 yes 5 ml 4 0 1 ml yes 5 ml 5 2 ml 0 no 5 ml 6 0 1 ml no 5 ml 7 0 0no 5 ml

Next, experiments were conducted to determine the effects of the type ofextraction solvent (method 1, whole broth), times used for extraction(method 1, hexane extraction, whole broth), and the amount of methanoladded prior to extraction (method 1, hexane extraction, whole broth).The results are presented in the Tables 17b-19 below.

TABLE 17b Solvent Farnesol (ng) Hexane 3273 Chloroform 2262 Ethylacetate 3549 N-heptane 3560 Hexadecane Not determined* Dodecane 3529Toluene 1055 Carbon tetrachloride 2023 Isobutyl Alcohol 2892 1-Octanol1814 *Solvent peak coeluted with farnesol, making quantification offarnesol impossible using this method.

TABLE 18 Vortex time (min) Farnesol (ng) 0 204 0.25 2794 0.5 2691 1.02894 1.5 3302 2.0 3348 2.5 3357 3.0 3545 3.5 3535

TABLE 19 Methanol volume (ml) Farnesol (ng) 0 3440 0.2 2820 0.4 3000 0.63440 0.8 3690 1.0 3730 1.2 4580 1.4 4350 1.6 4020 1.8 3880

Further work was done to optimize and simplify the extraction procedurefor farnesol, and to adapt the procedure for extraction of GG fromfermentation broths. This work resulted in the final extraction protocoldescribed below.

-   -   1. Whole fermentation broth is used undiluted or diluted        appropriately with water to a final volume of 2.0 ml in a 15 ml        teflon capped glass test tube.    -   2. 2.0 ml of hexane is added.    -   3. 2.0 ml of methanol is added.    -   4. The tube is vortexed at high speed for 3.5 minutes.    -   5. The sample is centrifuged at 2,800 rpm in a table top        centrifuge for 25-30 minutes to separate the phases.    -   6. The supernatant (organic phase) is removed and placed in a        2.0 ml teflon capped glass GC/MS vial for determination of        farnesol and GG by GC/MS.

Example 8

The following example demonstrates the effect of HMG CoA reductaseamplification on GG production by a strain over-expressing GGPPsynthase.

In this experiment, a strain was constructed that contained a singlecopy of a plasmid integrated into its genome that allows the cells toover-express GGPP synthase. The integrating plasmid is known aspSW35-42, and was constructed as follows. Plasmid pSW4A (see Example 5)was digested with BamHI to generate a 2199 bp fragment containing afusion of the ADH1 promoter, crtE coding sequence, and ADH1 terminator.This fragment was then ligated into BamHI digested YIp351 (Hill et al,1986, Yeast/E. coli Shuttle Vectors with Multiple Unique RestrictionSites, YEAST 2:163-167) to generate pSW35-42.

To construct a strain containing pSW35-42, this plasmid was digestedwith the restriction endonuclease BstEII to introduce a single cutwithin the LEU2 gene. The linearized plasmid was purified and used totransform strain SWE23-DL1 using the LiOAc method. Transformants inwhich pSW35-42 had integrated at the LEU2 locus were selected on mediumlacking leucine. One of the resulting transformed strains is referred toas SWE23-DL1::pSW35-42. This strain was then transformed with eitherYEp352 (an empty vector control, Hill et al, 1986, YEAST 2:163-167) orwith pRH124-31 (contains the catalytic domain of the HMG2 gene, seeExample 4), which resulted in strains SWE23-DL1::pSW35-42/YEp352 andSWE23-DL1::pSW35-42/pRH124, respectively. StrainSWE23-DL1::pSW35-42/YEp352 over-expresses GGPP synthase whileSWE23-DL1::pSW35-42/pRH124 over-expresses GGPP synthase and HMG CoAreductase.

Fermentation experiments of strains SWE23-DL1::pSW35-42/pRH124 andSWE23-DL1::pSW35-42/Yep352 were conducted to compare the GG productionby these two strains. The strains were tested for GG production in 1-Lfermentors using the protocol described in Example 1.H. The fermentationdata are presented in Table 20. This experiment shows that thefermentation of SWE23-DL1::pSW35-42/pRH124 accumulated more GG than thefermentation of SWE23-DL1::pSW35-42/Yep352, and demonstrates thatelevation of HMG CoA reductase activity in a strain designed to produceGG resulted in a further elevation of GG levels as compared to a similarstrain with un-amplified HMG CoA reductase.

TABLE 20 Dry Cell Weight Farnesol @192 hr @192 hr GG @192 hr Strain g/lg/l % dry wt. g/l % dry wt. SWE23-ΔL1::pSW35- 41.4 2.04 4.9 0.30 0.7242/Yep352 SWE23-ΔL1::pSW35- 44.0 3.27 7.4 0.60 1.36 42/pRH124

A second approach to achieve over-expression of HMG CoA reductase andGGPP synthase in the same strain was accomplished by cloning both genesinto a high copy-number yeast vector, and transforming this plasmid intoan erg9 mutant strain. The plasmid used for this purpose is referred toas pSW46-1, and this was constructed as follows. Plasmid pSW4A (seeExample 5) was digested with EcoRI and SacI and the 0.9 kb fragmentcontaining the crtE gene was purified and ligated into EcoRI, SacIdigested pADH313-956 to generate pSW38-10. Two regions of this plasmidwere deleted by digestion with restriction enzymes, filling the ends ofthe DNA using Pfu polymerase, and ligating to reform the circularplasmid. The regions deleted in this manner were between the NdeI andSpeI sites, and between the HindIII and PstI sites. The resultingplasmid is referred to as pSW43-5. Plasmid pSW43-5 was digested withBamHI, and the resulting 2199 bp fragment containing the ADH1 promoter,crtE gene, and ADH1 terminator (referred to as the ADHp/crtE/ADHtfragment) was purified and ligated into the BamHI site of YEp352. Theresulting plasmid is referred to as pSW44-17, and differs from pSW4A inthat it contains one instead of two PstI sites.

Plasmid pRH124-31 was digested with XbaI and PstI and the 3.2 kBfragment containing the GPD promoter, HMG2 catalytic domain gene, andthe PGK terminator (referred to as the GPDp/HMG2cat/PGKt fragment) wascloned into XbaI, PstI digested pSW44-17 to generate pSW46-1. Thisplasmid replicates to high copy number in yeast and contains both HMG2and crtE genes, providing for over-expression of HMG CoA reductase andGGPP synthase. Plasmid pSW46-1 was transformed into the erg9 mutantstrain SWE-DE91, and URA+ transformants were isolated. Several of theresulting transformants were tested in a shake flask experiment and werecompared to a strain that over-expresses only GGPP synthase(EMS9-23/pSW4A). The results from that experiment are presented in Table21, and demonstrate that higher levels of GG accumulate in strains thatover-express both HMG CoA reductase and GGPP synthase when compared to astrain that over-expresses only GGPP synthase.

TABLE 21 Dry Cell GG Wt. % Dry Strain Over-expressed Genes mg/ml mg/mlWt. SWE23- control 4.35 0.0007 0.02 ΔE91/YEp352 EMS9-23/pSW4A GGPPsynthase 2.82 0.0469 1.66 SWE23- GGPP synthase 3.45 0.0866 2.45ΔE91/pSW46-1 HMG CoA reductase

These data support the idea that over-expression of HMG CoA reductase ina strain that is capable of producing elevated levels of GG leads to afurther increase in the amount of GG produced.

Example 9

The following example describes the production of more stable strainsthat over-express HMG CoA reductase.

As described in Example 4, strains were constructed that over-expressedHMG CoA reductase due to the presence of high-copy number replicatingplasmids containing the HMG1cat or HMG2cat genes. Since replicatingplasmids can frequently be lost during cell division, this mitoticinstablity of the replicating plasmids can lead eventually to cultureswith a high proportion of cells containing only normal HMG CoA reductaselevels. In order to obtain more stable strains that over-expressed HMGCoA reductase, copies of the gene coding for the catalytic domain ofHMG2p were integrated into the genome of strain SW23B. This wasaccomplished by first constructing a plasmid that allowed for multipleintegrations of the HMG2 gene into the spacer region of the rRNA gene.This approach is modeled after the rDNA integration method described byLopes et al, 1989 (Lopes, T. S., Klootwijk, J., Veenstra, A. E., van derAar, P. C., van Heerikhuizen, H., Raue, H. A., and Planta, R. J. 1989.High-copy-number integration into the ribosomal DNA of Saccharomycescerevisiae: a new vector for high-level expression. Gene 79:199-206).

PCR was used to amplify two regions of the rDNA locus that lie in theintergenic region between the gene coding for the 35S rRNA and the genecoding for the 5S rDNA, referred to here as the rDNA spacer region. Theoligonucleotides used to amplify the first of the two rDNA spacer regionfragments contained sequences corresponding to bases #11161 to #11130and the reverse complement of #10311 to #10330 of Gene Bank Accession#Z73326. The oligonucleotide sequences are listed below. The lower caseletters are used to indicate bases that were altered or added to createrestriction endonuclease recognition sites, which are indicated inparentheses following the oligonucleotide sequence.

SEQ ID NO: 24: R6XbaI Lo 5′-tctagaGGCACCTGTCACTTTGGAAAAAAAATATACGC-3′(XbaI) SEQ ID NO: 25: R7SacII Up 5′-ccgcggGCCGGAAATGCTCTCTGTTC-3′(SacII)

The DNA fragment generated from PCR amplification using these twooligonucleotides is referred to herein as the R6/R7 fragment. The R6/7fragment generated by PCR was cloned initially into the plasmidpCR-Script to generate pSW48-1, then cut from this plasmid by digestionwith XbaI and SacII. The resulting 759 bp fragment was then cloned intoXbaI, SacII digested pBluescript SK− (Stratagene) to generate pSW49-1.

The oligonucleotides used to amplify the second rDNA spacer regionfragment contained sequences corresponding to bases #11247 to #11272 andthe reverse complement of #12054 to #12072 of Gene Bank Accession#Z73326. The sequences of these oligonucleotides are listed below.

SEQ ID NO: 26: R5Sa1 UpB 5′CACgtCgACCATTCAAACTTTACTAC-3′ (SalI)SEQ ID NO: 27: R4ApaI LoB 5′-GAGGGCccGGTCCAGACAT-3′ (ApaI)

The DNA fragment generated from PCR amplification using the twooligonucleotides listed above is referred to herein as the R4/R5fragment. The R4/R5 fragment generated by PCR was digested with ApaI andSalI, and ligated into ApaI, SalI digested pSW49-1 to generate pSW52-11.

PCR was used to amplify the TRP1 gene such that the gene carried anincomplete promoter. The incomplete promoter was intended to reduceexpression of the TRP1 gene, and thereby provide a means of selectionfor high copy number integration of the final expression cassette. Theoligonucleotides used to the TRP1 gene contained sequences correspondingto bases #53 to #73 and the reverse complement of #804 to #823 of GeneBank Accession #J01374. The oligonucleotide sequences are listed below.

SEQ ID NO: 28: TRP1 SmaUp 5′-cccgggTATTGAGCACGTGAGTATA CG-3′ (SmaI)SEQ ID NO: 29: TRP1 BamLo 5′-ggatccGGCAAGTGCACAAACAATA C-3′ (BamHI)

In the next step, pSW50-1 was digested with BamHI and SmaI, and theresulting 779 bp TRP1 fragment was purified. Plasmid pRH124-31 wasdigested with PstI and SmaI to generate a 3261 bp fragment containingthe GPD promoter, HMG2 cat gene, and the PGK terminator (referred to asthe GPDp/HMG2cat/PGKt fragment), and this fragment was purified. TheTRP1 fragment and the GPDp/HMG2cat/PGKt fragment were ligated into PstI,BamHI digested pSW52-11 in a three fragment ligation to generatepSW58-77 and pSW58-45.

The DNA fragment containing the R6/R7-TRP1-GPD p/HMG2cat/PGK t-R4/R5gene fusions was cut from pSW58-77 bp digestion with the restrictionendonucleases KpnI and SacI. The 5671 bp fragment corresponding to theintegrating construct was purified and used to transform strain SW23B.Transformants were selected on SCE-trp medium. Strains isolated in thismanner were screened for elevation of HMG CoA reductase activity and bySouthern blot analysis for elevation of HMG2 gene copy number. Strainswere identified that carried one copy of the integrating construct, twocopies of the integrating construct, three copies of the integratingconstruct, and eight copies of the integrating construct, and arereferred to as SW23B#19, SW23B#31, SW23B#40, and SW23B#74, respectively.Table 22 gives the specific activity of HMG CoA reductase as determinedby in vitro enzyme assays, and copy number of the HMG cat gene asdetermined by Southern blotting. The HMG CoA reductase assay waspreviously described (Quain, D. E. and Haslam, J. M. 1979. The Effectsof Catabolite Derepression on the Accumulation of Steryl Esters and theActivity of β-Hydroxymethylglutaryl-CoA Reductase in Saccharomycescerevisiae. J. Gen. Microbiol. 111:343-351).

TABLE 22 HMG CoA Reductase GPDp/HMG2cat/PGKt Strain nmol/min mgcopies/cell SW23B 5.26 0 SW23B#19 14.53 1 SW23B#31 37.47 2 SW23B#4050.33 3 SW23B#76 59.04 8

These strains require exogenously supplied leucine and uracil for growthdue to the leu2 and ura3 mutations in these strains. To eliminate theneed to supply these nutrients during fermentation experiments, thestrains were transformed with a plasmid, pTWM138, containing functionalcopies of URA3, LEU2, and TRP1. The presence of this plasmid in thesestrains allowed the strains to grow without leucine and uracilsupplementation.

Fermentation experiments were carried out to compare farnesol productionby these strains. Also included in this experiment was the strainSWE23-DE91/pRH124-31 which contains the GPDp/HMG2cat/PGKt gene fusion ona high copy number plasmid (see Example 4.B). The strains were testedfor farnesol production in 1-L fermentors using the protocol describedin Example 1.H, and the data from this experiment are presented in Table23.

TABLE 23 Genes Ferm Dry Cell Farnesol Amplified Time Weight % Dry Straincopy no. hr g/l g/l Cell Wt. SW23B/pTWM138 control 216 53.3 3.25 6.10SW23B#19/pTWM138 HMG2 cat 192 52.4 4.58 8.74 1 copy SW23B#74/pTWM138HMG2 cat 216 43.5 4.70 10.80 8 copies SW23B/pRH124-31 HMG2 cat 192 43.84.89 11.16 >20 copies

These data support the idea that strains with elevated levels of HMG CoAreductase produce more farnesol than a strain with normal levels of HMGCoA reductase. The data also indicate that a strain containing eightintegrated copies of the HMG2cat gene produce essentially as muchfarnesol as a strain that carries more than 20 extrachromosomal copiesof the HMG2cat gene as is the case with strain SW23B/pRH124-31. A straincontaining a single integrated copy of the HMG2 cat gene produced morefarnesol than the control strain, but slightly less than strains withmore copies of the HMG2cat gene. In addition, the strains containingelevated HMG CoA reductase levels accumulated mevalonate in the culturewhile the strains with normal levels of HMG CoA reductase did not. Thissuggests that a step downstream of HMG CoA reductase limits carbon fluxin the pathway once the HMG CoA reductase enzyme activity has beenelevated (see Example 10). Carbon flux through the pathway is restrictedby the activity of one of the enzymes downstream of HMG CoA reductaseresulting in mevalonate accumulation in the medium.

Example 10

This example shows the effects of over-expression of multiple isoprenoidpathway genes in a strain that has an erg9 mutation and elevated levelsof HMG CoA reductase.

As shown in Examples 4 and 9, elevation of HMG CoA reductase levels ledto higher carbon flux through the isoprenoid/sterol pathway.Over-expression of other isoprenoid pathway genes in strains containingamplified HMG CoA reductase may further increase carbon flux throughthis pathway. Amplification of isoprenoid pathway genes in strains thathave elevated levels of HMG CoA reductase as well as a defective erg9gene may result in further elevation of farnesol levels. Also,amplification of isoprenoid pathway genes may result in furtherelevation of GG levels in strains that have a defective erg9 gene andhave elevated levels of HMG CoA reductase and GGPP synthase.

To test these ideas, plasmids were constructed that allowed for theover-expression of multiple isoprenoid pathway genes. One of theplasmids provided for the over-expression of mevalonate kinase,phosphomevalonate kinase, and diphosphomevalonate decarboxylase, codedby the ERG12, ERGS, and ERG19 genes respectively. This plasmid isreferred to as pSW77-69, and was constructed from DNA fragments obtainedfrom a number of plasmids containing single isoprenoid pathway genes.The construction of those plasmids is described first.

PCR was used to amplify the ERG12 gene for cloning. The oligonucleotidesused to amplify the ERG12 gene contained sequences corresponding tobases #2827 to #2846 and the reverse complement of #5247 to #5229 ofGene Bank Accession #Z49809. The sequences of the two oligonucleotidesare given below. The lower case letters are used for bases that werealtered or added to create restriction endonuclease recognition sites,which are indicated in parentheses following the oligonucleotidesequence.

SEQ ID NO: 30: E12.4SN 5′-CCAAATATAACtCGAGCTTTG-3 (XhoI) SEQ ID NO: 31:E12.SALI3 5′-GCAAAGTcCaCCACCGCAG-3 (SalI)

The ERG12 gene was amplified using the two oligonucleotides E12.4SN andE12.5ALI3 and genomic DNA from strain ATCC 28383. The resulting DNA wascloned into pCR-Script at the SfrI site to generate pSW68.

The oligonucleotides used to amplify the ERG19 gene contained sequencescorresponding to bases #911 to #937 of Gene Bank Accession #Z71656 andthe reverse complement of bases #1930 to #1962 of Gene Bank Accession#Z71658. The sequences of the two oligonucleotides are given below.

SEQ ID NO: 32: E19 SMAI 5′-GCCACGTGCCCcCGGGTTTCTCTAGCC-3′ (SmaI)SEQ ID NO:33: E19 SACI3 5′-GGAAAAGagCtCGATAATTATTGATGATAGATC-3′ (SacI)

The ERG19 gene was amplified using the two oligonucleotides E19 SMAI,E19 SACI3, and genomic DNA from strain ATCC 28383. The resulting DNA wascloned into pCR-Script at the SfrI site to generate pSW69.

The oligonucleotides used to amplify the ERG8 gene contained sequencescorresponding to bases #2729 to #2749 and the reverse complement of#5177 to #5196 of Gene Bank Accession #Z49939. The sequences of the twooligonucleotides are given below.

SEQ ID NO: 34: E8.2135N 5′-CCGTTTTGGATccTAGATCAG-3′ (BamHI)SEQ ID NO: 35: E8SMAI3 5′-gttcccGGGTTATTGTCCTGCATTTG-3′(SmaI)

The ERG8 gene was amplified using the two oligonucleotides E8.2135N,E8SMAI3, and genomic DNA from strain ATCC 28383. The resulting DNA wascloned into pCR-Script at the SfrI site to generate pSW71.

Plasmid pSW71 was digested with BamHI and SmaI, and the resulting 2459bp fragment containing the ERG8 gene was purified. Plasmid pSW69-3 wasdigested with SmaI and SacI, and the resulting 2239 bp fragmentcontaining the ERG19 gene was purified. The high copy number yeastvector YEp352 (containing a URA3 selection marker) was digested withBamHI and SacI and purified. The three purified DNA fragments wereligated together to generate pSW76-11.

Next, pSW68 was digested with SalI and XhoI and the resulting 2400 bpband containing the ERG12 gene was purified and ligated into SalIdigested pSW76-11 to generate pSW77-69. This plasmid contains ERG12,ERG8, and ERG19, and provides for over-expression of mevalonate kinase,phosphomevalonate kinase, and diphosphomevalonate decarboxylase.

Another plasmid provided for the over-expression of mevalonate kinase,phospho-mevalonate kinase, diphosphomevalonate decarboxylase, and IPPisomerase, coded by ERG12, ERG8, ERG19, and IDI1 genes respectively.This plasmid is referred to as pSW78-68, and was constructed as follows.The IDI1 gene was amplified by PCR for cloning. The oligonucleotidesused to amplify the IDI1 gene contained sequences corresponding to bases#19577 to #19604 and the reverse complement of #17477 to #17502 of GeneBank Accession #U43503. The sequences of the two oligonucleotides aregiven below.

SEQ ID NO: 36: ISO SACI5 5′-AAGAGctcATCTGATAATAGATCAAG CG-3′ (SacI)SEQ ID NO: 37: ISO SACI3 5′-AGGAGCTCAACGACAATAAATGGCTG-3′ (SacI)

The IDI1 gene was amplified using the two oligonucleotides ISO SACI5,ISOSACI3, and genomic DNA from strain ATCC 28383. The resulting DNA wascloned into pCR-Script at the SfrI site to generate pSW73. Plasmid pSW73was digested with SacI and the resulting 2117 bp fragment containing theIDI1 gene was ligated with Sad digested pSW77-69 to generate pSW78-68.This plasmid contains ERG12, ERG8, ERG19, and IDI1, and provides forover-expression of mevalonate kinase, phosphomevalonate kinase,diphosphomevalonate decarboxylase, and IPP isomerase.

Another plasmid provided for the over-expression of acetoacetyl CoAthiolase and HMG CoA synthase, coded by the ERG10 and ERG13 genes,respectively. This plasmid is referred to as pSW79-29, and wasconstructed as follows.

PCR was used to amplify the ERG13 and ERG10 genes for cloning. Theoligonucleotides used to amplify the ERG13 gene contained sequencescorresponding to the reverse complement of bases #21104 to #21127 andbases #18270 to #18292 of Gene Bank Accession #Z50178. The sequences ofthe two oligonucleotides are given below.

SEQ ID NO: 38: E13 XBAI5 5′-GTCCTctAGATCTTGAATGAAATC-3′(XbaI)SEQ ID NO: 39: E13 SACI3 5′-CTTTGAGCtcGTACAAGAAGCAG-3′ (SacI)

The ERG13 gene was amplified using the two oligonucleotides E13 XBAI5,E13 SACI3, and genomic DNA from strain ATCC 28383. The resulting DNA wascloned into pCR-Script at the SfrI site to generate pSW72.

The oligonucleotides used to amplify the ERG10 gene contained sequencescorresponding to bases #4073 to #4093 and the reverse complement of#6542 to #6565 of Gene Bank Accession #U36624. The sequences of the twooligonucleotides are given below.

SEQ ID NO: 40: E10 HIND 5′-CTAAGCttTGCGCCCGTGAAG-3′ (HindIII)SEQ ID NO: 41: E10 XBAI3-2 5′-GTTCTAGAAGTTTTCAAAGCAGAG-3′ (XbaI)

The ERG10 gene was amplified using the two oligonucleotides E10 HIND5,E10 XBAI3-2, and genomic DNA from strain ATCC 28383. The resulting DNAwas cloned into pCR-Script at the SfrI site to generate pSW70.

Plasmid pSW70 was digested with XbaI and HindIII, and the resulting 2482bp fragment containing the ERG10 gene was purified. Plasmid pSW72-4 wasdigested with XbaI and SacI, and the resulting 2850 bp fragmentcontaining the ERG13 gene was purified. The two purified DNA fragmentswere then ligated together with SacI, HindIII digested YEp351 (LEU2selectable marker) to generate pSW79-30. This plasmid contains the ERG13and ERG10 genes and allows for the over-expression of acetoacetyl CoAthiolase and HMG CoA synthase.

Strain SW23B#74 (contains eight integrated copies of the HMG2 cat gene,see Example 9) was transformed with pSW77-69 and pSW78-68, andtransformants were selected on SCE-ura medium. The resulting strains arereferred to as SW23B#74/pSW77-69 and SW23B#74/pSW78-68, respectively.These strains require added leucine for growth due to the leu2 mutation.To eliminate the need to supplement these strains with leucine duringfermentation experiments, they were transformed with a linear fragmentof DNA containing a functional LEU2 gene, and LEU+ transformants wereisolated. These strains are referred to as SW23B#74L/pSW77-69 andSW23B#74L/pSW78-68, respectively.

SW23B#74 was also transformed with pSW79-30, and transformants wereselected on SCE-leu medium. SW23B#74/pSW79-30 requires uracil since itcontains a mutation in the ura3 gene. To eliminate the need tosupplement these strains with uracil during fermentation experiments,this strain was transformed with a linear fragment of DNA containing afunctional URA3 gene, and URA+ transformants were isolated. This strainis referred to as SW23B#74U/pSW79-30.

SW23B#74 was also transformed with both pSW78-68 and pSW79-30, andtransformants were isolated that were capable of growing on SCE-ura,-leumedium indicating that the transformants contained both plasmids. Theresulting strain is referred to as SW23B#74/pSW78-68/pSW79-30.

Fermentation experiments were carried out with the strains describedabove to examine the effect of these gene amplifications on farnesolproduction. The strains were tested in 1-L fermentors using the protocoldescribed in Example 1.H, and data from these experiments are presentedin Table 24.

TABLE 24 Ferm. Dry Cell Farnesol Mevalonate Genes Time Weight % Dry %Dry Strain Amplified (hr) g/l g/l Wt. g/l Wt. SW23B#74/ HMG2cat 215 45.54.95 10.88 0.2 0.4 pTWM138 SW23B#74L/ HMG2cat, 287 41.6 4.67 11.23 0 0pSW77-69 ERG8, ERG12, ERG19, SW23B#74L/ HMG2cat, 215 46.4 4.87 10.50 0 0pSW78-68 ERG8, ERG12, ERG19, IDI1 SW23B#74U/ HMG2cat, 215 46.1 4.6310.04 5.4 11.7 pSW79-30 ERG10, ERG13

These data show that a strain which over-expresses HMG CoA reductaseaccumulates the isoprenoid pathway intermediate mevalonate in theculture medium. Since mevalonate accumulation is not observed in strainswith normal levels of HMG CoA reductase, this demonstrates that carbonflux through the isoprenoid pathway has been increased by HMG CoAreductase amplification to the point where another step subsequent toHMG CoA reductase limits the conversion of pathway intermediates.Furthermore, over-expression of the first three enzymes in theisoprenoid pathway, namely acetoacetyl CoA thiolase, HMG CoA synthase,and HMG CoA reductase (coded by ERG 13, ERG10, and HMG2cat,respectively) led to even higher accumulation of mevalonate in themedium. This demonstrates that carbon flux into the isoprenoid pathwayhas been increased further by amplification of the first three steps,and emphasizes that one of the enzymes downstream of HMG CoA reductaselimits conversion of isoprenoid pathway intermediates. Since mevalonateserves as a precursor to farnesol and GG, the modifications describedabove can be used to increase carbon flux into the isoprenoid pathway,and, if the mevalonate can be more efficiently metabolized, can lead toincreased farnesol and GG accumulation.

In regard to this last point, enzymes downstream of HMG CoA reductasewere amplified in a strain that over-expressed HMG CoA reductase. Theenzymes amplified were HMG CoA reductase, mevalonate kinase,phosphomevalonate kinase, diphosphomevalonate decarboxylase, and IPPisomerase (coded by HMG2cat, ERG12, ERGS, ERG19, and IDI1,respectively). These strains accumulated less mevalonate when comparedto strains over-expressing only HMG CoA reductase, indicating that theincreased flux through the isoprenoid pathway resulting from HMG CoAreductase over-expression was accommodated by elevated levels of thedownstream enzymes. Although elevated farnesol levels were not observedin the experiment described above, this may be due to the low level ofmevalonate available for conversion. However, these data suggest thatamplification of one or more of the enzymes downstream of HMG CoAreductase in a strain with amplified levels of the first three pathwayenzymes may lead to increased accumulation of farnesol and GG since fluxto mevalonate is greatly increased in these strains. It is likely thatover-expression of one or more genes downstream of HMG CoA reductase ina strain that over-expresses acetoacetyl CoA thiolase, HMG CoA synthase,and HMG CoA reductase will lead to significant increases in farnesol andGG accumulation.

Example 11

This example describes an experiment in which squalene synthase isblocked by zaragozic acid in a strain with an intact and functionalsterol pathway, including a functional ERG9 gene, and the effect of thissqualene synthase inhibitor on farnesol and GG accumulation is observed.

Zaragozic acids are a family of compounds that act as potent inhibitorsof squalene synthase (Bergstrom, J. D., Dufresne, C., Bills, G. F.,Nallin-Omstead, M., and Byrne, K. 1995. Discovery, Biosynthesis, andMechanism of Action of the Zaragozic Acids: Potent Inhibitors ofSqualene Synthase. Ann. Rev. Microbiol. 49:607-639). They are capable ofinhibiting cholesterol biosynthesis in mammals and ergosterolbiosynthesis in fungi. Since ergosterol is necessary for fungal cellgrowth, zaragozic acids are potent fungicidal compounds.

Strain SWE23-E9 (ura3, sue; see EXAMPLE 1.G) was used in this experimentbecause it contained the sue mutation that provided for the uptake ofsterols under aerobic conditions. This allowed the yeast to grow inergosterol-supplemented medium when squalene synthase was blocked byzaragozic acid. A strain without the sue mutation would not be able totake up sterols, including ergosterol, from the medium, and thereforecould not grow in the presence of zaragozic acid.

SWE23-E9 containing either an empty vector (Yep352) or plasmids thatallow for the over-expression of HMG Co A reductase (pRH124-31), GGPPsynthase (pSW4A), or both HMG Co A reductase and GGPP synthase (pSW46-1)were tested in shake flasks by first growing the strains in SCE-uramedium for 48 hr to select for the plasmids. Samples of these cultureswere then inoculated into YPDE medium or YPDE medium containingzaragozic acid at 100 μg/ml. The YPDE (+/−zaragozic acid) cultures wereincubated at 30° C. for 48 hr, then analyzed for dry cell weight andfarnesol accumulation. The data from this experiment are shown in Table25. All of the cultures treated with zaragozic acid exhibited elevatedaccumulation of farnesol relative to the untreated cultures. GGaccumulation was not measured in this experiment.

TABLE 25 Zaragozic Dry Cell Farnesol Acid Weight % Dry Strain @100 μg/mmg/ml μg/ml Wt. SWE23-E9/Yep 352 + 7.65 43 0.56 SWE23-E9/Yep352 − 9.72 00 SWE23-E9/pRH124-31 + 7.88 11 0.14 SWE23-E9/pRH124-31 − 9.89 0 0SWE23-E9/pSW4A + 7.03 45 0.64 SWE23-E9/pSW4A − 9.05 1 0SWE23-E9/pSW46-1 + 7.09 51 0.72 SWE23-E9/pSW46-1 − 8.54 0 0

In a separate experiment, SWE23-E9 containing either an empty vector(Yep352) or a plasmid that allows for over-expression of both HMG Co Areductase and GGPP synthase (pSW46-1) were tested in shake flasks byfirst growing the strains in SCE-ura medium for 48 hr to select for theplasmids. Samples of these cultures were then inoculated into YPDEmedium or YPDE medium containing zaragozic acid at 100 mg/ml. The YPDE(+/−zaragozic acid) cultures were incubated at 30° C. for 72 hr, thenanalyzed for dry cell weight and GG. The data are presented in Table 26.The culture of SWE23-E9/pSW46-1 treated with zaragozic acid exhibited ahigher level of GG as compared to the same strain grown in the absenceof zaragozic acid. These experiments demonstrate that strains withoutmutations in the sterol biosynthetic pathway can be induced toaccumulate farnesol by blocking the pathway using a squalene synthaseinhibitor. The amount of farnesol or GG accumulated by the zaragozicacid treated cultures was much less than the amount accumulated bystrains with completely defective erg9 genes indicating that a geneticblock of squalene synthase is more effective than a block induced by asqualene synthase inhibitor.

TABLE 26 Zaragozic Dry Cell Acid Weight GG Strain @100 μg/ml mg/ml μg/ml% Dry Wt. SWE23-E9/Yep352 + 7.17 0 0 SWE23-E9/Yep352 − 9.25 0 0SWE23-E9/pSW46-1 + 6.68 17.8 0.27 SWE23-E9/pSW46-1 − 8.41 6.7 0.08

Example 12

An alternative pathway leading to FPP has been described in a number ofbacteria and plants, and is referred to as the non-mevalonate or Rohmerpathway (Eisenreich, W., Schwarz, M., Cartayrade, A., Arigoni, D., Zenk,M. H., and Bacher, A., 1998. The Deoxyxylulose Phosphate Pathway ofTerpenoid Biosynthesis in Plants and Microorganisms. Chem. Biol. 5:R221-233. Paseshnichenko, V. A., 1998. A New Alternative Non-MevalonatePathway for isoprenoid Biosynthesis in Eubacteria and Plants.Biochemistry (Mosc) 63:139-148). Some of the enzymes of thenon-mevalonate pathway and their genes have been identified and studiedin E. coli, and these include the deoxyxylulose-5-phosphate synthase,deoxyxylulose-5-phosphate reductase, IPP isomerase, and FPP synthase,coded by the dxr, dxs, idi, and ispA genes, respectively.

In order to construct strains of E. coli that accumulate elevated levelsof farnesol, plasmids were constructed for the over-expression in E.coli of the genes listed above. In some cases, additional genes adjacentto the known isoprenoid pathway gene were included in the cloned DNA.

The idi gene coding for IPP isomerase was PCR amplified using the twooligonucleotides listed below and genomic DNA isolated from E. colistrain W3110. The oligonucleotides used to amplify the idi genecontained sequences corresponding to bases #42309 to #42335 and thereverse complement of #43699 to #43726 of Gene Bank Accession #U28375.The lower case letters are used for bases that were altered to createrestriction endonuclease recognition sites, which are indicated inparentheses following the oligonucleotide sequence. A natural EcoRI sitewas used in VE145-5.

SEQ ID NO: 42: VE145-5 5′-GGGATTCGAATTCTGTCGCGCTGTAAC-3′ (EcoRI)SEQ ID NO: 43: VE146-3 5′-TGggATCcGTAACGGCTTTAGCGAGC TG-3′ (BamHI)

The idi PCR product was digested with EcoRI and BamHI, and ligated intoEcoRI, BamHI digested pUC19. The resulting clone is referred to aspHS-0182.

The dxr gene was PCR amplified using the two oligonucleotides listedbelow and genomic DNA isolated from E. coli strain W3110. Theoligonucleotides used to amplify a region of DNA which included the frr,dxr, and yaeS genes contained sequences corresponding to bases #2131 to#2150 and the reverse complement of #5297 to #5315 of Gene BankAccession #D83536.

SEQ ID NO: 44: DXR17.SN GATTCCGCGTAAGgATCcCG (BamHI) SEQ ID NO: 45:DXR3179.ASN CCAGCATctAGACCACCAG (XbaI)

The resulting PCR product was digested with BamHI and XbaI, and ligatedinto BamHI, XbaI digested pHS-0182 to generate pHS-O182R.

The ispA and dxs genes appear to be part of an operon that possiblyinclude two other genes referred to as xseB and yajO. PCR was used toamplify a region of DNA which included xseB, ispA, dxs, and yajO usingthe oligonucleotides listed below and genomic DNA isolated from E. colistrain W3110. The oligonucleotides contained sequences corresponding tobases #4157 to #4183 and the reverse complement of bases #8938 to #48956of Gene Bank Accession #AE000148.

SEQ ID NO: 46: DXS.5619P 5′-gactgcagCTGGCCGCTGGGTATTCTGTCGTAGTT-3′(PstI) SEQ ID NO: 47:DXS.820X 5′-gatctagaTCACGCGTACGCAGAAGGTTTTG C-3′ (XbaI)

The resulting PCR product was digested with XbaI and PstI and ligated toXbaI, PstI digested pUC19 to generate pHS-dxs.

The DNA fragment containing the dxs gene will be cut from pHS-dxs usingXbaI and PstI, and ligated into XbaI, PstI digested pHS-0182R togenerate a plasmid that contains dxs, dxr, ispA, and idi. The resultingplasmid will be transformed into E. coli, and a transformed straincontaining the plasmid will be tested for production of farnesol inshake flask and fermentation experiments.

In order to construct strains of E. coli that accumulate elevated levelsof GG, a plasmid was constructed for the over-expression in E. coli ofGGPP synthase from Erwinia herbicola. The crtE gene coding for GGPPsynthase was amplified by PCR using genomic DNA isolated from Erwiniaherbicola strain Eho10 and the following two oligonucleotides. Theoligonucleotides contained sequences corresponding to bases #3513 to#3531 and the reverse complement of bases #4459 to #4481 of Gene BankAccession #M87280.

SEQ ID NO: 48: Eho10E Up 5′-gaattcCAATTCAGCGGGTAACCTT-3′ (EcoRI)SEQ ID NO: 49: Eho10E Lo 5′-aagcttTGCTTGAACCCAAAAGGGCGGTA-3′ (HindIII)

The resulting PCR product was digested with EcoRI and HindIII, andligated into pET24d(+) (Novagen) so that expression of the ctrE gene wascontrolled by the T7 promoter. The resulting plasmid is referred to aspKL19-63. This plasmid can be transformed into E. coli strains such asBL21 (DE3) (available from Novagen) which contains an IPTG induciblegene coding for T7 polymerase. This allows IPTG induction of the crtEgene in this strain. The T7 promoter/crtE gene fusion can be cut frompKL19-63 using BglII and HindIII, and ligated into BamHI, HindIIIdigested pACYC184 (Accession #X06403) to construct a plasmid relying onchloramphenicol resistance for selection. This plasmid contains the pl5Aorigin of replication and would be compatible with plasmids containingthe ColE1 origin of replication such as the clones carrying the E. coliisoprenoid pathway genes described above. These latter plasmids conferampicillin resistance, and so E. coli transformants can be obtained thatcarry both the crtE plasmid and the plasmid containing the dxs, dxr,idi, and ispA genes by transforming E. coli with both plasmids andselecting for resistance to chloramphenicol and ampicillin. Therefore,strains of E. coli BL21 (DE3) will be obtained that contain plasmids forover-expression deoxyxylulose-5-phosphate synthase,deoxyxylulose-5-phosphate reductoisomerase, IPP isomerase, FPP synthase,and GGPP synthase. These strains will be tested for production of GG inshake flask and fermentation experiments.

Example 13

The following example describes the construction of strains ofSaccharomyces cerevisiae that are engineered to produce elevated levelsof farnesol and GG by expressing enzymes corresponding to thenon-mevalonate pathway enzymes.

Biosynthesis of IPP occurs in many bacteria and plants starting frompyruvate and glyceraldehyde-3- and proceeding through a series ofenzymatic steps known as the non-mevalonate pathway, and includes theenzymes deoxyxylulose-5-phosphate synthase and deoxyxylulose-5-phosphatereductoisomerase. The compound resulting from these two enzymatic stepsis 2-C-methyl-D-erythritol 4-phosphate (also known as MEP), which isfurther metabolized by unknown enzymes to yield IPP. Plasmids areconstructed that express the E. coli dxs and dxr genes in yeast byfusing the coding regions of those genes to promoter elements that allowfor expression in yeast. The dxs and dxs genes are amplified by PCR witholigonucleotides that include restriction sites to allow cloning intoyeast expression vectors which contain promoters such as the ADH1promoter, PGK promoter, or GPD promoter. The two gene fusions are thencombined into a single plasmid by subcloning one gene fusion into theother plasmid. The resulting plasmid containing both genes is thentransformed into an erg9 mutant of yeast. The resulting strain iscapable of synthesizing MEP, which may be further metabolized to IPP byendogenous enzymes capable of carrying out the desired reactions to IPP.This capability may lead to increased accumulation of IPP, which maycause the cells to accumulate elevated levels of farnesol through theaction of the endogenous IPP isomerase and FPP synthase enzymes. If thisis done in a strain that also over-expresses GGPP synthase, thenelevated levels of GG may accumulate in these strains.

Example 14

This example describes the construction of a strain of Saccharomycescerevisiae that over-expresses a mutated ERG20 gene coding for an FPPsynthase enzyme exhibiting altered product specificity. Unlike strainsover-expressing wild-type FPP synthase, strains expressing the mutatedFPP synthase accumulated more GG than farnesol.

Comparison of the amino acid sequences of FPP synthases from a varietyof organisms revealed several highly conserved domains including twoaspartate rich domains, which are essential for catalytic activity.Researchers have reported that mutations effecting the amino acidlocated at the fifth position before the first aspartate rich domain canalter the product specificity of the enzyme such that the enzyme readilycatalyzes the formation of GGPP as well as FPP. This has been shown forboth avian and Bacillus stearothermophilus FPP synthases (Tarshis, L.C., Proteau, P. J., Kellogg, B. A., Sacchettini, J. C., Poulter, C. D.1996. Regulation of Product Chain Length by Isoprenyl DiphosphateSynthases. Proc. Natl. Acad. Sci. USA 93:15018-15023; Ohnuma, S.,Narita, K., Nakazawa, T., Ishida, C., Takeuchi, Y., Ohto, C., andNichino, T. 1996. A Role of the Amino Acid Residue Located on the FifthPosition Before the First Aspartate-rich Motif of Farnesyl DiphosphateSynthase on Determination of the Final Product. J. Biol. Chem.271:30748-30754; U.S. Pat. No. 5,766,911). In the case of the Bacillusenzyme, the amino acid in the fifth position before the first aspartaterich domain was changed from tyrosine to serine. The avian enzymecontains a phenylalanine in this position. It is thought that aromaticamino acids in that position block extension of the isoprenyl chainbeyond fifteen carbons, and that the restriction can be alleviated byreplacing the phenylalanine with a less-bulky amino acid such as serine.

As in the Bacillus FPP synthases mentioned above, the FPP synthase fromSaccharomyces cerevisiae has a tyrosine at the fifth position before thefirst aspartate rich domain. Site directed mutagenesis was used to alterthe sequence of the ERG20 gene to code for a serine instead of atyrosine at that position. The method used to introduce the mutationrelied on PCR amplification of the entire pJMB 19-31 plasmid usingmutagenic oligonucleotides that introduced the desired mutation, and isoutlined in the instruction booklet for the QuikChange Site-DirectedMutagenesis Kit (Stratagene). The oligonucleotides contained sequencescorresponding to bases #1063 to #1097 and the reverse complement ofbases #1063 to #1097 of Gene Bank Accession #J05091. The sequences ofthe oligonucleotides are given below, with the changes indicated bysmall case letters. An alteration of A to T was made at position #1084to change the encoded amino acid from tyrosine to serine. Also, analteration of T to C was made at position #1074 to create a new PstIsite, which allowed for rapid identification of the mutant gene. Thislatter mutation was silent in that it did not change the encoded aminoacid.

SEQ ID NO: 50: Y95S-SN 5′-GCATTGAGTTGcTGCAGGCTTcCTTCTTGGTCGCC-3′SEQ ID NO: 51: Y95S-ASN 5′-GGCGACCAAGAAGgAAGCCTGCAgCAACTCAATGC-3′

The two oligonucleotides were used to amplify pJMB19-31, and theresulting PCR product was ligated to itself to reform the circularplasmid, except that ERG20 gene now contained the desired mutation. Theresulting plasmid is referred to as pHS31.Y95S, and this plasmid wastransformed into the erg9 mutant strain SWE23-DE91 to formSWE23-DE91/pHS31.Y95S. Shake flask experiments were carried out tocompare farnesol and GG production by strains over-expressing thewild-type ERG20 gene and the mutated ERG20 gene. Strains were grownovernight in SCE-ura, and this was used to inoculate flasks containingYPDE medium. These cultures were grown at 30° C. for 72 hr, thenharvested for dry cell weight and isoprenoid analysis. The data fromthis experiment is presented in Table 27.

TABLE 27 Dry Cell Farnesol GG FPP Wt % Dry % Dry Strain Synthase mg/mlmg/ml Wt. mg/ml Wt. SWE23-DE91/ Wild type 4.7 0.22 4.76 0.05 1.1JMB19-31 SWE23-DE91/ Tyr to Ser 4.9 0.01 0.2 0.13 2.7 pHS31.Y95S Mutant

These data show that the farnesol:GG ratio is dramatically altered byover-expression of the tyr to ser mutant of the ERG20 gene in an erg9mutant strain. The strain over-expressing the mutant ERG20 geneexhibited proportionally more GG than the strain over-expressing thewild-type ERG20 gene. In addition, the total amount of GG produced bythe strain over-expressing the mutant ERG20 was higher while the totalamount of farnesol was lower as compared to the strain over-expressingthe wild-type ERG20 gene. The decrease in farnesol level was notcompletely reflected in the GG pool, suggesting that some of the FPP maybe converted to other compounds besides GG. Alternatively, it ispossible that feedback inhibition plays a role in regulating the amountof GG accumulated by these strains.

The foregoing description of the invention has been presented forpurposes of illustration and description. Furthermore, the descriptionis not intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with the aboveteachings, and the skill or knowledge of the relevant art, are withinthe scope of the present invention. The embodiments describedhereinabove are further intended to explain best modes known ofpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with thevarious modifications required by the particular applications or uses ofthe invention. It is intended that the appended claims be construed toinclude alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A recombinant yeast that is capable of making anisoprenoid compound, the recombinant yeast comprising: an endogenousmevalonate pathway comprising (i) an enzyme that converts acetyl-CoA toacetoacetyl-CoA, (ii) an enzyme that converts acetoacetyl-CoA tohydroxymethylglutaryl-CoA, (iii) an enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid, (iv) an enzyme thatconverts mevalonic acid to phosphomevalonic acid, (v) an enzyme thatconverts phosphomevalonic acid to phosphomevalonate, and (vi) an enzymethat converts phosphomevalonate to isopentenyl pyrophosphate; and atleast one heterologous nucleic acid sequence encoding an enzyme thatconverts hydroxymethylglutaryl-CoA to mevalonic acid; at least oneheterologous nucleic acid sequence encoding an enzyme that convertsmevalonic acid to phosphomevalonic acid; at least one heterologousnucleic acid sequence encoding an enzyme that converts phosphomevalonicacid to phosphomevalonate; and at least one heterologous nucleic acidsequence encoding an enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; wherein carbon flow through the mevalonatepathway is increased in the recombinant yeast compared to a yeast thatdoes not comprise a heterologous nucleic acid sequence encoding theenzyme that converts hydroxymethylglutaryl-CoA to mevalonic acid.
 2. Therecombinant yeast of claim 1, wherein the enzyme that convertsacetyl-CoA to acetoacetyl-CoA is a thiolase.
 3. The recombinant yeast ofclaim 1, wherein the enzyme that converts acetoacetyl-CoA tohydroxymethylglutaryl-CoA is an HMG Co A synthase (HMGS).
 4. Therecombinant yeast of claim 1, wherein the enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid is an HMG Co A reductase(HMGR).
 5. The recombinant yeast of claim 1, wherein the enzyme thatconverts mevalonic acid to phosphomevalonic acid is a mevalonate kinase(MK).
 6. The recombinant yeast of claim 1, wherein the enzyme thatconverts phosphomevalonic acid to phosphomevalonate is aphosphomevalonate kinase (PMK).
 7. The recombinant yeast of claim 1,wherein the enzyme that converts phosphomevalonate to isopentenylpyrophosphate is a phosphomevalonate decarboxylase (PMD).
 8. Therecombinant yeast of claim 1, wherein at least one of (i) the enzymethat converts hydroxymethylglutaryl-CoA to mevalonic acid, (ii) theenzyme that converts mevalonic acid to phosphomevalonic acid, (iii) theenzyme that converts phosphomevalonic acid to phosphomevalonate, and(iv) the enzyme that converts phosphomevalonate to isopentenylpyrophosphate is under inducible control.
 9. The recombinant yeast ofclaim 1, wherein (i) the enzyme that converts hydroxymethylglutaryl-CoAto mevalonic acid, (ii) the enzyme that converts mevalonic acid tophosphomevalonic acid, (iii) the enzyme that converts phosphomevalonicacid to phosphomevalonate, and (iv) the enzyme that convertsphosphomevalonate to isopentenyl pyrophosphate is under induciblecontrol.
 10. The recombinant yeast of claim 1, wherein at least one ofthe heterologous nucleic acid sequences selected from the groupconsisting of the at least one heterologous nucleic acid sequenceencoding the enzyme that converts hydroxymethylglutaryl-CoA to mevalonicacid; the least one heterologous nucleic acid sequence encoding theenzyme that converts mevalonic acid to phosphomevalonic acid; the atleast one heterologous nucleic acid sequence encoding an enzyme thatconverts phosphomevalonic acid to phosphomevalonate; and the at leastone heterologous nucleic acid sequence encoding the enzyme that convertsphosphomevalonate to isopentenyl pyrophosphate; is integrated into thegenome.
 11. The recombinant yeast of claim 1, wherein each of theheterologous nucleic acid sequences in the group consisting of the atleast one heterologous nucleic acid sequence encoding the enzyme thatconverts hydroxymethylglutaryl-CoA to mevalonic acid; the least oneheterologous nucleic acid sequence encoding the enzyme that convertsmevalonic acid to phosphomevalonic acid; the at least one heterologousnucleic acid sequence encoding the enzyme that converts phosphomevalonicacid to phosphomevalonate; and the at least one heterologous nucleicacid sequence encoding the enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; are integrated into the genome.
 12. Therecombinant yeast of claim 1, wherein the yeast comprises more than oneheterologous nucleic acid sequence encoding an enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid.
 13. The recombinant yeastof claim 1, wherein the yeast further comprises at least oneheterologous nucleic acid sequence encoding an enzyme that convertsacetoacetyl-CoA to hydroxymethylglutaryl-CoA.
 14. The recombinant yeastof claim 13, wherein the enzyme that converts acetoacetyl-CoA tohydroxymethylglutaryl-CoA is an HMGS.
 15. The recombinant yeast of claim1, wherein the yeast is Saccharomyces cerevisiae.
 16. The recombinantyeast of claim 1, wherein the enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid is a bacterial enzyme. 17.The recombinant yeast of claim 1, wherein the enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid is a yeast enzyme.
 18. Therecombinant yeast of claim 1, wherein the enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid, the enzyme that convertsmevalonic acid to phosphomevalonic acid, the enzyme that convertsphosphomevalonic acid to phosphomevalonate, and the enzyme that convertsphosphomevalonate to isopentenyl pyrophosphate are each eukaryotic. 19.The recombinant yeast of claim 1, wherein the enzyme that convertshydroxymethylglutaryl-CoA to mevalonic acid, the enzyme that convertsmevalonic acid to phosphomevalonic acid, the enzyme that convertsphosphomevalonic acid to phosphomevalonate, and the enzyme that convertsphosphomevalonate to isopentenyl pyrophosphate are each yeast enzymes.20. The recombinant yeast of claim 1, wherein each enzyme in the groupconsisting of: the enzyme that converts hydroxymethylglutaryl-CoA tomevalonic acid, the enzyme that converts mevalonic acid tophosphomevalonic acid, the enzyme that converts phosphomevalonic acid tophosphomevalonate, and the enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; is encoded on a different heterologousnucleic acid molecule.
 21. The recombinant yeast of claim 1, wherein atleast two enzymes selected from the group consisting of: the enzyme thatconverts hydroxymethylglutaryl-CoA to mevalonic acid, the enzyme thatconverts mevalonic acid to phosphomevalonic acid, the enzyme thatconverts phosphomevalonic acid to phosphomevalonate, and the enzyme thatconverts phosphomevalonate to isopentenyl pyrophosphate; are encoded onthe same heterologous nucleic acid molecule.
 22. The recombinant yeastof claim 1, wherein at least three enzymes selected from the groupconsisting of: the enzyme that converts hydroxymethylglutaryl-CoA tomevalonic acid, the enzyme that converts mevalonic acid tophosphomevalonic acid, the enzyme that converts phosphomevalonic acid tophosphomevalonate, and the enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; are encoded on the same heterologous nucleicacid molecule.
 23. The recombinant yeast of claim 1, wherein the enzymethat converts hydroxymethylglutaryl-CoA to mevalonic acid, the enzymethat converts mevalonic acid to phosphomevalonic acid, the enzyme thatconverts phosphomevalonic acid to phosphomevalonate, and the enzyme thatconverts phosphomevalonate to isopentenyl pyrophosphate are encoded onthe same heterologous nucleic acid molecule.
 24. A recombinant yeastthat is capable of making an isoprenoid compound, the recombinant yeastcomprising: an endogenous mevalonate pathway comprising (i) an enzymethat converts acetyl-CoA to acetoacetyl-CoA, (ii) an enzyme thatconverts acetoacetyl-CoA to hydroxymethylglutaryl-CoA, (iii) an enzymethat converts hydroxymethylglutaryl-CoA to mevalonic acid, (iv) anenzyme that converts mevalonic acid to phosphomevalonic acid, (v) anenzyme that converts phosphomevalonic acid to phosphomevalonate, and(vi) an enzyme that converts phosphomevalonate to isopentenylpyrophosphate; and at least one heterologous nucleic acid sequenceencoding an enzyme that converts hydroxymethylglutaryl-CoA to mevalonicacid; at least one heterologous nucleic acid sequence encoding an enzymethat converts mevalonic acid to phosphomevalonic acid; at least oneheterologous nucleic acid sequence encoding an enzyme that convertsphosphomevalonic acid to phosphomevalonate; and at least oneheterologous nucleic acid sequence encoding an enzyme that convertsphosphomevalonate to isopentenyl pyrophosphate; wherein an activityselected from the group consisting of conversion ofhydroxymethylglutaryl-CoA to mevalonic acid, (ii) conversion ofmevalonic acid to phosphomevalonic acid, (iii) conversion ofphosphomevalonic acid to phosphomevalonate, and (iv) conversion ofphosphomevalonate to isopentenyl pyrophosphate is increased in therecombinant yeast compared to a yeast that does not comprise the atleast one heterologous nucleic acid sequence encoding an enzyme thatconverts hydroxymethylglutaryl-CoA to mevalonic acid.
 25. Therecombinant yeast of claim 24, wherein each enzyme in the groupconsisting of: the enzyme that converts hydroxymethylglutaryl-CoA tomevalonic acid, the enzyme that converts mevalonic acid tophosphomevalonic acid, the enzyme that converts phosphomevalonic acid tophosphomevalonate, and the enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; is encoded on a different heterologousnucleic acid molecule.
 26. The recombinant yeast of claim 24, wherein atleast two enzymes selected from the group consisting of: the enzyme thatconverts hydroxymethylglutaryl-CoA to mevalonic acid, the enzyme thatconverts mevalonic acid to phosphomevalonic acid, the enzyme thatconverts phosphomevalonic acid to phosphomevalonate, and the enzyme thatconverts phosphomevalonate to isopentenyl pyrophosphate; are encoded onthe same heterologous nucleic acid molecule.
 27. The recombinant yeastof claim 24, wherein at least three enzymes selected from the groupconsisting of: the enzyme that converts hydroxymethylglutaryl-CoA tomevalonic acid, the enzyme that converts mevalonic acid tophosphomevalonic acid, the enzyme that converts phosphomevalonic acid tophosphomevalonate, and the enzyme that converts phosphomevalonate toisopentenyl pyrophosphate; are encoded on the same heterologous nucleicacid molecule.
 28. The recombinant yeast of claim 24, wherein the enzymethat converts hydroxymethylglutaryl-CoA to mevalonic acid, the enzymethat converts mevalonic acid to phosphomevalonic acid, the enzyme thatconverts phosphomevalonic acid to phosphomevalonate, and the enzyme thatconverts phosphomevalonate to isopentenyl pyrophosphate are encoded onthe same heterologous nucleic acid molecule.